virus and representatives of the two other genera of the Parvoviridae. The densonucleosis viruses (DNVs) form a unique group among parvoviruses in that they.
Vol. 37, No. 1
JOURNAL OF VIROLOGY, Jan. 1981, p. 17-23 0022-538X/81/010017-07$02.00/0
Biochemical, Biophysical, and Biological Properties of Densonucleosis Virus (Parvovirus) III. Common Sequences of Structural Proteins EDOUARD KURSTAK* Comparative Virology Research Group, Faculty ofMedicine, Universite de Montreal, Montreal, Quebec, PETER TIJSSEN
AND
Canada
Densonucleosis virus cannot code for its four structural proteins if each of them has a unique sequence. The objective of the present investigation, therefore, was to establish whether: (i) the viral genome contains overlapping genes; (ii) the virus incorporates host proteins; or (iii) one of the structural proteins is a dimer. Two independent methods were employed for this purpose. First, the viral proteins, solubilized in sodium dodecyl sulfate, were purified after dansylation and were analyzed by peptide mapping, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, an enzyme-linked immunosorbent assay was developed for a comparative analysis of the viral proteins solubilized by sodium dodecyl sulfate. It was demonstrated with both techniques that densonucleosis virus has four unique structural proteins, all with extensive sequence homologies. Moreover, all structural proteins contained intraprotein, but no interprotein, disulfide linkages. These results indicated similarities between densonucleosis virus and representatives of the two other genera of the Parvoviridae.
teins remain suitable substrates for a limited proteolysis; and (iii) the electrophoretic separation of these proteins (or fragments) can be monitored at any time under UV light. In the previous paper (23), we presented some peptide maps of DNV, and we demonstrated, using this technique, that unrelated proteins have strikingly different peptide maps. The purified viral proteins (solubilized in SDS and dansylated) are equally suitable for an immunological analysis. The ELISA technique was adapted for this purpose by following the observation that antiserum prepared against purified, untreated virus could, under certain conditions, react with denatured proteins fixed to a polystyrene plastic. This antigen-antibody reaction was shown to be inhibited by an earlier absorption of the antiserum with the corresponding soluble SDS antigen. Consequently, antigenic relationships among the viral proteins could be established by the competitive inhibition of the immunological reaction, using homologous or heterologous viral antigens. Moreover, dansylation of the proteins used for the coating or for the absorption did not affect the serological response. It was suspected (25) that p98 was a dimer of p49 as: (i) the molecular weight of p98 was exactly double that of p49; (ii) the percentage of p98 in the virion fluctuated greatly; and (iii) in a similar virus, AAV type 3, it was reported that
The densonucleosis viruses (DNVs) form a unique group among parvoviruses in that they have both autonomous replication and the complementary DNA strands separately encapsidated (2, 10, 14, 16, 17). It was shown previously (25) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that DNV from Galleria mellonella larvae contains four structural proteins. The total molecular weight of these proteins (p49, p59, p69, and p98) exceeds the coding capacity of the viral genome (25). This phenomenon, observed for almost all of the parvoviruses studied in this respect, might be explained either by an incorporation of host proteins into the virion or by the presence of overlapping genes in the viral DNA. Molecular similarities among the structural proteins have been reported for the minute virus of mice (22) and for an adenoassociated virus (AAV) (4, 9, 15). We will demonstrate here by two independent methods, peptide mapping and a modified enzyme-linked immunosorbent assay (ELISA), that the four structural proteins of DNV also have sequence homologies. We have described previously (23) the parameters and operational details of the method used here for the peptide mapping of the viral proteins. It is based on the observation that: (i) dansylated SDS proteins can be easily purified by preparative electrophoresis; (ii) these pro17
18
TIJSSEN AND KURSTAK
the fourth protein band can be dissociated with urea into monomers (8). However, attempts to dissociate the p98 into p49 failed (25). We will demonstrate in this paper that p98 is, in fact, a monomer. MATERLALS AND METHODS Extraction and purification of the virus. Procedures used previously (24) for the extraction and purification of the virus have been modified slightly. Larvae were either inoculated intraperitoneally in the seventh instar (25) or naturally infected in the early instars by introducing one or two animals killed by DNV into the culture. The dead larvae were putrefied for several days at room temperature in Hanks solution. This preparation was diluted (1:1) with a 0.025 M sodium phosphate buffer solution (pH 7.5) before the purification of the virus (this buffer solution was used throughout the purification procedure). Homogenization and fat extraction (24) were followed by centrifugation at 16,000 rpm for 75 min in a Sorvall RC2-B centrifuge equipped with an SS-34 rotor. The pellet was suspended and centrifuged once more. The two supernatants were then pooled and subjected to differential centrifugation. The virus was pelleted at 40,000 rpm (A-321 rotor, IEC-B centrifuge) for 75 min, suspended in the buffer solution, and centrifuged at 20,000 rpm for 30 min. The supernatant was centrifuged at 40,000 rpm for 75 min, and the pellet was recovered as partially purified virus. This virus preparation was then centrifuged to equilibrium in CsCl gradients by the method described previously (25). Separation and isolation of the viral proteins. DNV, at a concentration of 3 mg/ml in a 0.1 M Trisacetate buffer solution (pH 8.2), was solubilized and dansylated as described previously (23). The labeled viral proteins were separated on SDSpolyacrylamide gels (diameter, 11 mm) in a Buchler electrophoresis apparatus by the method of Laemmli (12). The dansyl-labeled proteins, as shown below, comigrated with their unlabeled counterparts. The separation of the proteins during electrophoresis could be readily monitored under long-wave UV light (365 nm) through the glass tubes. Electrophoresis was stopped after a maximum resolution among the viral proteins was attained. Gel slices containing the protein bands were cut from the gel with a thin metal wire. The corresponding slices were packed together into another electrophoresis tube, and the proteins were eluted at 10 mA per gel into a dialysis bag until no fluorescent material remained in the gel. The eluted protein preparations were dialyzed against either the peptide mapping buffer or the ELISA buffer. The purity of the protein was confirmed by SDS-PAGE (12). Peptide mapping of the structural proteins. The enzymes Staphylococcus aureus protease (Miles Laboratories, Inc.), chymotrypsin (Miles Laboratories, Inc.), and papain (Sigma Chemical Co.) were suspended in a 0.125 M Tris-hydrochloride buffer solution (pH 6.8) containing 10% glycerol. A 5-,ul amount of enzyme suspension was added to 27 ,lI of the viral protein samples (0.3 to 0.5 mg of protein per ml in 0.125 M Tris-hydrochloride buffer solution [pH 6.81
J. VIROL.
containing 10%o glycerol, 0.5% SDS, and 0.001% bromophenol blue) to give a final concentration of 50, 50, and 1 ,ug of the respective enzymes, per ml. The samples were incubated for 30 min at 37°C. The enzymatic reaction was stopped by the addition of 5 ,ul of a 30% 2-mercaptoethanol-25% SDS solution and boiling for 2 min in a water bath. The peptide fragments were analyzed by SDSPAGE (10 to 20% gradient) (23). The generation of the peptide maps could be followed by illuminating the gel with long-wave UV light. Modified enzyme-linked immunosorbent assays. The conventional ELISA technique (3) was adapted for a comparative analysis of the purified, viral proteins solubilized in SDS. The following solutions were prepared: (i) the conjugate of peroxidase linked to goat anti-rabbit immunoglobulin G antibodies (0.002 optical density unit per ml at 280 nm; the 401 nm/280 nm absorbance ratio of the conjugate was 0.26) as described elsewhere (11); (ii) phosphate-buffered saline (PBS; 8.0 g of NaCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4. 12H20, 0.2 g of KCI, and 0.5 g of polysorbate 20 [Tween 20] per liter); (iii) PBS-ST (PBS containing 0.1% SDS and 0.5% Triton X-100); and (iv) a stock solution of each of the four dansylated, structural proteins in 5.0% SDS. The concentrations of these protein stock solutions were determined by the method of Hess et al. (7). The SDS was removed from the proteins by ionpair extraction (6). The antigen was suspended, and the suspension was diluted further with a 0.05 M sodium carbonate (pH 9.6) buffer solution until a protein concentration of 5 ,ug/ml was obtained. For each of the purified proteins, a separate plate (eight rows of six wells) was sensitized overnight at 4°C. The plates were subsequently washed three times for 5 min with PBS. Samples of rabbit antiserum against DNV, prepared by the method of Harboe and Ingild (5), were absorbed separately by each of the four proteins. For this purpose, 5.25-volumes of antiserum, diluted 21 times with PBS, were added to 1 volume of protein sample (20 ,ug/ml in PBS-ST) and dialyzed overnight against PBS at room temperature. Antiserum absorbed similarly with bovine serum albumin (or only PBS-ST) served as a control. The absorbed and control sera were serially diluted twofold with PBS and tested on each of the four plates sensitized with p49, p59, p69, or p98 (see Fig. 3). The incubation and detection by the immunoperoxidase technique were performed as described elsewhere (3). Detection of disulfide linkages. The presence of inter- or intraprotein disulfide linkages was studied by the addition of 2-mercaptoethanol to the virus samples containing 1% SDS, or by its omission from these samples, and subsequent analysis by SDS-PAGE. The discontinuous SDS-PAGE procedure (13) was used. The samples contained 1% SDS, 0.125 M Tris-hydrochloride buffer solution (pH 6.8), and 10% glycerol. Other samples contained, in addition to these reagents, 1% 2-mercaptoethanol. The samples were dialyzed extensively against the sample buffer solution (without 2-mercaptoethanol) unless stated otherwise. Electrophoresis was carried out at 20 mA per gel at room temperature in a vertical slab gel apparatus (model
19 220; Bio-Rad Laboratories). The gels were stained have been expected if p98 were indeed a dimer COMMON SEQUENCES OF DNV PROTEINS
VOL. 37, 1981
with 0.25% Coomassie blue 250G in a solution of 9% acetic acid and 45% methanol and destained by diffusion in a solution of 7.5% acetic acid and 5% methanol.
of p49. Peptide mapping. The purified proteins were subjected to a limited proteolysis by various enzymes and analyzed on SDS-polyacrylamide gradient slabs. The peptide maps obRESULTS tained for the four viral proteins were very simPurification of the virus. Present modifi- ilar (Fig. 2) if the same enzyme was used. Differcations of the virus purification procedures al- ent enzymes produced completely different lowed a significant increase (as expressed in maps. It is of interest that the fragments obabsorbancy units at 260 nm per infected larva) tained after the digestion of p49 were also found in the yield of DNV. Naturally infected larvae for the other three viral proteins of higher moand inoculated larvae yielded 1.5 and 0.5 to 1.2 lecular weight. The latter, however, contained absorbancy units of the virus at 260 nm per extra bands accounting for a difference in moleclarva, respectively (average of five experiments, ular weight. For example, the S. aureus protease about 500 larvae for each). digest of viral proteins p59, p69, and p98 conIsolation of the viral proteins. A pilot ex- tained almost all of the fragments of p49 (Fig. 2a periment, in which a sample of dansylated pro- and d). The S. aureus protease digest of p69 teins was mixed with an equal quantity of unla- contained, besides an extra fragment when combeled proteins and subjected to SDS-PAGE, pared with p59, the extra fragment obtained in showed that, after staining with Coomassie blue, the p59 digest when the latter was compared the protein profile was unaltered. Since dansyl with p49. The p98 digest contained all p69 fragchloride had no effect on the mobility of the ments. Extra bands were also present in the viral SDS proteins, this reagent is suitable for peptide maps of the structural proteins of higher the purification of those proteins. molecular weight digested by the other enzymes The viral proteins were eluted from the gel (Fig. 2b and e, papain; Fig. 2c and f, chymotrypslices and recovered in the dialysis bags, each of sin). which contained one of the four viral proteins. The fluorescent maps were similar to the maps Although no exact recovery determinations have obtained after staining the same slabs with Coobeen attempted, the absence of fluorescence in massie blue (Fig. 2d, e, and f). The higher senthe slices after elution showed that losses during sitivity of the fluorescence method, as compared these procedures were negligible. The purity of with the conventional Coomassie blue staining, the isolated proteins was tested by SDS-PAGE is particularly evident with the peptide frag(Fig. 1). Figure 1 also shows that the p49 and ments of lower molecular weight. The enzymes the p98 eluates did not give rise to p98 and p49 were not labeled with dansyl chloride; therefore, bands, respectively, after SDS-PAGE, as might they were not visible in the fluorescent maps. ELISA techniques. The- antiserum had a titer of 25,600 against DNV when tested with the conventional, indirect ELISA technique (3), whereas extracts from noninfected larvae did not react. The modified ELISA technique demonstrated that all viral structural proteins react with the antiserum prepared against complete DNV par-
ticles (Fig. 3, series A, C, E, and G). The reaction of the antisera with the p49 protein (i.e., the reaction of this protein with its specific antibody; Fig. 3, plate p49) was inhibited efficiently by the absorption of the antiserum not only with p49 (Fig. 3, series B), but also with p59, p69, and p98 (Fig. 3, series D, F, and H, respectively). This competitive inhibition was also evident in the reverse situation, in which another protein was FIG. 1. Isolation of the structural proteins. The coated to the plate and the antibodies for that viral proteins, dissociated by SDS and 2-mercaptoadsorbed to soluble p49 (Fig. 3, ethanol (lane 1), were dansylated and purified after protein were series p59, p69, and p98). The reduction B, plates separation by SDS-PAGE. The purity ofp98 (lane 2), p49 (lane 3), p59 (lane 4), and p69 (lane 5) was checked of the titer after absorption of the sera with by re-electrophoresis on an SDS-polyacrylamide slab. homologous proteins was at least 16 to 64 times when tested on plates in which p49, p59, or p69 The proteins of lane were not dansylated. 1
20
TIJSSEN AND KURSTAK
.satgS
J. VIROL.
12X 345s 6 7 8- 9 10
-0D
2-3 4 5 _,,J,, :
+
,;
.
7:
W __
-;
-0:0 _l
ff.:
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7
*
:E
_l ;u .
iS
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i-
,> w l, mBX E
|!
,:.... X
FIG. 2. Peptide maps of the viral proteins. (a), (b), and (c) were photographed directly after electrophoresis, whereas (d), (e), and (f) represent the same slabs after staining with Coomassie blue. Lanes 1, 2, 3, and 4 were loaded with the purified proteins (p49, p59, p69, and p98, respectively). Lanes 6, 7, 8, and 9 contained the viral proteins, in the same order, digested by one of the proteases (S. aureus protease in [a] and [d]; papain in [b] and [el; chymotrypsin in [c] and [fi). The corresponding proteases, run in lane 10, were not visible in the dansylation map. The arrowheads indicate extra bands in a map of a protein with a higher molecular weight when compared with the preceding maps. The circles indicate bands visible in the p49 and p69 maps but not in the p59 map. The dissociated virus was subjected to electrophoresis in lane 5. The front band in lane 5 contains dansylation by-products.
had been coated. However, the reduction of the titer of the antisera, tested on a plate sensitized with p98, was significantly less (approximately four times) for each of the heterologous proteins but stronger for the homologous protein (p98). The absorption of the antiserum by p98 when tested on the p49 plate (Fig. 3, series H) was less efficient when compared with the absorption by p49. This was probably due to the use of the same quantity (in weight) of absorbing protein; hence, there were twice as many p49 as p98
molecules. This effect was also visible elsewhere (e.g., absorption by p69 and testing on the p49 plate or absorption by p98 and testing on the p59 and the p69 plates). In control tests, bovine serum albumin (native or SDS denatured) was not able to reduce the titers obtained. These experiments demonstrated that (i) the four structural proteins have antigenic determinants in common and (ii) p98, in addition to the antigenic determinants shared with the other proteins, contained unique sequences.
VOL. 37, 1981
COMMON SEQUENCES OF DNV PROTEINS
p49
1 234 5 6 AB
D5zy
21
1 23 45 6 p 59 A
E F G_
E
H_qlq 1 234 5 6
p6 A D
1 2 34 5 6 p98 A
cX
_
D] E_ Fi
E F
Gx H
,Mf
H
FIG. 3. ELISA of the isolated, viral proteins. Each of the isolated proteins was coated to a different plate (p49, p59, p69, and p98 plates). The DNV antiserum, diluted 25, 50, 100, 200, 400, and 800 times in columns 1, 2, 3, 4, 5, and 6, respectively, reacted with each of the coated proteins (series A; repeated in series C, E, and G). The titer of the DNV antiserum, after absorption by p49, was significantly reduced when tested on any of the viral proteins (series B), but not after absorption by unrelated proteins (data not shown). The other viral proteins also reduced the antiserum titer significantly for both the homologous and the heterologous proteins (series D, antiserum absorbed by p59; series F, antiserum absorbed by p69; series H, antiserum absorbed by p98).
Presence of disulfide linkages. Interprotein disulfide linkages in the virion are absent since the omission of 2-mercaptoethanol did not affect the dissociation of the virion by SDS (Fig. 4). However, all viral proteins contain intraprotein S bridges, as the omission of 2-mercaptoethanol resulted in increased mobilities of the viral proteins in SDS-polyacrylamide gels (Fig. 4). The omission of the dialysis step after dissociation caused the 2-mercaptoethanol to diffuse from the slots containing the reduced preparation to the slots containing the virus dissociated in SDS only (Fig. 4). The proteins of the latter were reduced at the side adjacent to the slot containing 2-mercaptoethanol, whereas the protein at the other side of the slot migrated as nonreduced protein.
DISCUSSION The main result of the present study was the observation that all four structural proteins of DNV possess common sequences.
The peptide maps indicated that p98 is a monomer and not, as previously suspected (see above), a dimer of p49, since the supplementary
fragments of p59 and p69, in addition to the p49 fragments, were found in the p98 digest. The same conclusion could be drawn from the findings obtained with the modified ELISA technique. The reaction of the antigen with its specific antibody was most efficiently inhibited by the absorption of the serum with the same antigen, especially for p69 and p98. The titer of the same antiserum sample, absorbed with p49, p59, or p69, was most efficiently reduced when tested on plates coated with one of these three proteins. The reduction of this titer was significantly less when tested on plates coated with p98. However, antiserum absorbed with the same quantity of p98 had a strongly reduced titer when tested on plates sensitized with p49, p59, or p69. These results showed that p98 has unique sequences and is not a dimer of p49. The sequence homology among the DNV polypeptides also explains
22
TIJSSEN AND KURSTAK
FIG. 4. Disulfide linkages in the viral proteins. The virions were efficiently dissociated by SDS (lane 1). However, the electrophoretic mobility of each of the proteins was reduced after reduction of the proteins by 2-mercaptoethanol and dialysis to remove 2mercaptoethanol (lane 2). The omission of the dialysis step after the reduction (lane 4) allowed 2-mercaptoethanol to diffuse from this lane to the neighboring one (lane 3) containing virus treated with SDS only and thus reduced the proteins nearest lane 4.
how the DNV genome, with a coding capacity of 200,000 (25), is able to code for four structural proteins with a total molecular weight of 275,000. The exact overlap of the proteins will be established by current research in this laboratory, using CNBr fragmentation techniques on the viral proteins and the protein fragments obtained by limited proteolysis with enzymes. The modified ELISA technique proved to be a sensitive method for the detection of common determinants on proteins. In recent experiments (data not shown), this technique was simplified further by coating one plate with the absorbing proteins, diluting the antiserum into the wells of this plate, and, after a 20-h incubation, transferring each (diluted) antiserum sample to another plate on which the protein to be tested was coated. The detection of bound antibodies on both plates by immunoenzymatic methods revealed a possible relationship. Results thus obtained for the DNV proteins were not different from the ELISA results described here. The hydrophobic regions of many extracellu-
J. VIROL.
lar proteins, in sharp contrast to intracellular proteins, often contain disulfide bonds, conferring additional stability to these proteins (1). Covalent disulfide bonds between the viral proteins of the DNV particle were shown to be absent. However, disulfide linkages are present within all of the structural proteins (another common feature of these proteins). These S-S bridges could play an important structural role in these proteins as there is a considerable restriction in rotation about the S-S bond (equivalent to approximately 40 to 80 kJ [13]). It was observed in this laboratory that some qualitative parameters, such as the sharpness of the protein bands in polyacrylamide gels, and the percentage of intact S-S bridges were closely correlated (data not shown). It is of interest, in this respect, that putrefaction (a practice widely used in insect virology [20]) of infected larvae and subsequent purification in Hanks solution yielded homogeneous preparations of the virus, since the viral proteins contained more than 85% intact disulfide bonds. The disadvantage of homogenization as compared with putrefaction of the infected larvae is possibly caused by an adsorption of the virus to the host tissues during homogenization. The buffer used in the purification system has also been modified. Possible effects by bivalent ions on the virion have been noted earlier (24). The reports of Buller and Rose (4) and Salo and Mayor (18) state, in contrast to the report of Johnson et al. (9), that the concentrations of the AAV-induced polypeptides relative to one another remain approximately constant during the infectious cycle. Unpublished data in our laboratory demonstrated important stoichiometric differences in the DNV preparations from different origins; e.g., the virus used for the experiments described in this paper had only about 40% p49 (Fig. 1 and 4) in contrast to the "normal" 70 to 80% (25). This does not seem to support the hypothesis that the molar proportions of these proteins do not change during parvovirus replication (4). For the autonomous parvoviruses, i.e., the genus Parvovirus, a varying stoichiometry of the structural proteins has been reported (21), and a precursor has been described for the minute virus of mice (also based on peptide mapping [22]). This has also been suggested for AAV (based on kinetic studies and immunochemical relationships [9]). It was suggested in the latter study that a nonstructural viral protein (molecular weight, 120,000) might be processed into the structural proteins. This was not confirmed by pulse-chase experiments which indicated that the AAV polypeptides are generated at the level of protein synthesis (4). It was reported (4) that the argi-
VOL. 37, 1981
COMMON SEQUENCES OF DNV PROTEINS
nine analog L-canavanine blocks the appearance of the major structural AAV peptide and one of the two smaller nonstructural polypeptides. This, in turn, was not confirmed by Salo and Mayor (18), who were unable to detect any of the nonstructural proteins described by either Johnson et al. (9) or Buller and Rose (4). However, they observed a diffuse band of 40,000 daltons. Unfortunately, no tissue cultures are as yet available for pulse-chase studies of the DNVs (14). It is tempting to speculate that the expression of the DNV genome in young larvae is regulated differently from that in older larvae and thus explain the completely different stoichiometry of the structural proteins in the virus obtained from the young larvae when compared with the virus obtained from last instar larvae. The role of RNA splicing in cell differentiation has been suggested in other systems (19) and might also operate as a regulatory factor for the development of successive larval instars. Research is being carried out to resolve these transcription and splicing questions. LITERATURE CITED 1. Anfinsen, C. B. 1972. The formation and stabilization of protein structure. Biochem. J. 128:737-749. 2. Bachmann, P. A., M. D. Hoggan, E. Kurstak, J. L. Melnick, H. G. Pereira, P. Tattersall, and C. Vago. 1979. Parvoviridae: second report. Intervirology 11:248254. 3. Bidwell, D. C., A. A. Buck, H. J. Dresfeld, B. Enders, J. Haworth, G. Huldt, N. H. Kent, C. Kirsten, P. Mattern, E. J. Ruitenberg, and A. Voller. 1976. The enzyme-linked immunosorbent assay (ELISA). Bull. W.H.O. 54:129-139. 4. Buller, R. M. L., and J. A. Rose. 1978. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J. Virol. 25:331-3,38. 5. Harboe, N., and A. Ingild. 1973. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand. J. Immunol. 2(Suppl. 1):161-164. 6. Henderson, L. E., S. Oroszlan, and W. Koningsberg. 1979. A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction. Anal. Biochem. 93:153-157. 7. Hess, H. H., M. B. Lees, and J. E. Derr. 1978. A linear Lowry-Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins. Anal. Biochem. 85: 295-300. 8. Johnson, F. B., H. L. Ozer, and M. D. Hoggan. 1971. Structural proteins of adenovirus-associated virus type 3. J. Virol. 8:860-863.
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9. Johnson, F. B., T. A. Thomson, P. A. Taylor, and D. A. Vlazny. 1977. Molecular similarities among the adenovirus-associated virus polypeptides and evidence for a precursor protein. Virology 82:1-13. 10. Kurstak, E., P. Tijssen, and S. Garzon. 1977. Densonucleosis viruses (Parvoviridae), p. 67-91. In K. Maramorosch (ed.), Ultrastructure in biological systems, an atlas, vol. 7. Academic Press, Inc., New York. 11. Kurstak, E., P. Tijssen, and C. Kurstak. 1977. Immunoperoxidase techniques in diagnostic virology and research: principles and applications, p. 403-448. In E. Kurstak and C. Kurstak (ed.), Comparative diagnosis of viral diseases, vol. 2, part B. Academic Press, Inc., New York. 12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680-685. 13. Liu, T.-Y. 1977. The role of sulfur in proteins, p. 240-402. In H. Neurath, R. L. Hill, and C. L. Boeder (ed.), The proteins, vol. 3. Academic Press, Inc., New York. 14. Longworth, J. F. 1978. Small isometric viruses of invertebrates. Adv. Virus Res. 23:103-157. 15. Lubeck, M. D., H. M. Lee, M. D. Hoggan, and F. B. Johnson. 1979. Adenovirus-associated virus structural protein sequence homology. J. Gen. Virol. 45:209-216. 16. Mayor, H. D., and E. Kurstak. 1974. Viruses with separately encapsidated complementary strands, p. 55-78. In E. Kurstak and K. Maramorosch (ed.), Viruses, evolution and cancer. Academic Press, Inc., New York. 17. Rose, J. A. 1974. Parvovirus reproduction, p. 1-61. In H. Fraenkel-Conrat and R. Wagner (ed.), Comprehensive virology, vol. 3. Plenum Publishing Corp., New York. 18. Salo, R. J., and H. D. Mayor. 1979. Adenovirus-associated virus polypeptides synthesized in cells coinfected with either adenovirus or herpesvirus. Virology 93:237245. 19. Segal, S., A. J. Levine, and G. Khoury. 1979. Evidence for nonspliced SV40 RNA in undifferentiated murine teratocarcinoma stem cells Nature (London) 280:335338. 20. Smith, K. M. 1967. Insect virology. Academic Press, Inc., New York. 21. Tattersall, P., P. J. Cawte, A. J. Shatkin, and D. C. Ward. 1976. Three structural polypeptides coded for by minute virus of mice, a parvovirus. J. Virol. 20:273289. 22. Tattersall, P., A. J. Shatkin, and D. C. Ward. 1977. Sequence homology between the structural polypeptides of minute virus of mice. J. Mol. Biol. 111:375-394. 23. Tijssen, P., and E. Kurstak. 1979. A simple and sensitive method for the purification and peptide mapping of proteins solubilized from densonucleosis virus with sodium dodecyl sulfate. Anal. Biochem. 99:97-104. 24. Tijssen, P., T. Tijssen-van der Slikke, and E. Kurstak. 1977. Biochemical, biophysical, and biological properties of densonucleosis virus (parvovirus). II. Two types of infectious virions. J. Virol. 21:225-231. 25. Tijssen, P., J. van den Hurk, and E. Kurstak. 1976. Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins. J. Virol. 17: 686-691.
