NUCLEIC ACID FROM AN ADENO-ASSOCIATED VIRUS:CHEMICAL AND PHYSICAL STUDIES*

BY JAMES A. ROSE, M. DAVID HOGGAN, AND AARON J. SHATKIN

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES,

NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND

Communicated by Robert J. Huebner, May 24, 1966

Virus particles approximately 22 mgu in diameter have been purified from severalhuman and simian adenovirus preparations.1 2 The purified particles are anti-genically unrelated to adenovirus and require the addition of adenovirus for replica-tion to levels detectable by electron microscopy.1' 2 Since these particles dependon the presence of adenovirus for their detectable replication, they have been calledadeno-associated virus (AAV).1AAV found in simian adenovirus type 15 preparations has been reported

to contain double-stranded DNA on the basis of acridine orange staining.1 How-ever, AAV nucleic acid has not been further characterized. The present work wasundertaken to define chemical and physical properties of nucleic acid from the AAVfound in a preparation of human adenovirus type 7 strain LL E46+ (E46+)2, 3and to compare these properties with those of E46+ DNA. The results indicatethat this AAV, now designated AAV serotype 1,2 contains double-stranded DNAwith a base composition and molecular weight differing significantly from E46+DNA.

Experimental Procedure.-Materials: A KB cell line was provided by Dr. M. Green; primarymonolayer cultures of human embryonic kidney (HEK) cells were purchased from Flow Labora-tories. Optical grade CsCl was obtained from Harshaw Chemical Co., 5'-deoxynucleotides fromCalbiochem, thymidine-H3 from Nuclear-Chicago and Schwarz BioResearch, Inc., uridine-H3from New England Nuclear Corp., and P32-orthophosphate from International Chemical andNuclear Corp. Snake venom phosphodiesterase, electrophoretically purified pancreatic DNase I,2X crystallized papain, and crystallized-lyophilized trypsin were purchased from WorthingtonBiochemical Corp.

Viruses: Preparation and assays of stock pools of adenovirus type 7 strain LL E46+ and thesubstrain E46- are described elsewhere.2 One E46+ stock contained 102-103 AAV particles/-adenovirus particle and had an adenovirus infectivity titer of 106 TCID50/ml in HEK cells. Asecond E46+ stock, passaged three times in the presence of AAV antiserum, and the E46- stockcontained no demonstrable AAV by electron microscopy, complement fixation (CF), and immuno-fluorescent antibody testing. These stocks had adenovirus infectivity titers of 108 TCID5o/mland 109 TCID5o/ml, respectively, in HEK cells. Both E46 + stocks, with or without AAV, inducedadenovirus and SV40 tumor antigens in HEK and African green monkey kidney (AGMK) cells.4

Virus production and purification: HEK monolayer cultures in 32-oz bottles were grown inEagle's medium (BME) containing 10% calf serum. For infection, cultures were washed threetimes with BME, and 40 ml of BME containing 2% agammaglobulinic, heated (560C for 30 min)calf serum (Hyland Laboratories) was added. Cultures were then inoculated with E46 stocks at-0.01 TCID5o/cell and incubated at 370C until typical adenovirus cytopathic effects developed(3-4 days). The cells were scraped into the medium, collected by centrifugation at 1100 X g, andsuspended in tris buffer, 0.01 M pH 8.1. Released virus was also recovered by centrifuging thesupernatant fluid at 78,000 X g for 3 hr. The pellet was combined with the cells and frozen andthawed six times. All subsequent operations were carried out at 0-40C. The suspension waspipetted vigorously ten times through a narrow bore pipette and tris buffer added to a final volumeof 1.5 ml for each 32-oz culture bottle. After homogenization with an equal volume of redistilledgenetron 113, the aqueous extract was adjusted to a density of 1.39 gm/cm3 by addition of solidCsCl and centrifuged at 33,000 rpm for 40 hr in the SW-39 rotor of the Spinco model L centrifuge.

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Equilibrium density gradients were fractionated by collecting drops through a puncture in thebottom of the centrifuge tube. The density of selected fractions was determined from the refrac-tive index measured with an Abbe refractometer. Adenovirus was purified by recentrifugingcombined fractions (1.33-1.37 gm/cm3) at 33,000 rpm for 24 hr in tris-buffered CsCl at an averagedensity of 1.35 gm/cm3. Adenovirus type 2 (adenoid 6) was produced in KB cells and purifiedas previously described,5 6 except that the last purification step was carried out in CsCl equilibriumdensity gradients. AAV was purified by centrifuging combined fractions (1.38-1.42 gm/cm3)twice more at 33,000 rpm for 40 hr in tris-buffered CsCl at an average density of 1.39 gm/cm3.The final virus bands were dialyzed against SSC (0.15 M NaCl, 0.015 M sodium citrate). Allpreparations were examined by electron microscopy and tested by CF.2 Purified E46+ had thecapacity to induce both adenovirus and SV40 tumor antigens in HEK and AGMK cells.

Extraction and purification of viral DNA: 0-Mercaptoethanol (0.025 ml) and 0.015 ml papain(7 enzyme units) were added to each ml of dialyzed virus suspension. For adenovirus DNA, themixture was incubated 5 hr at 370C with a second addition of papain after 2.5 hr. A 0.1-vol of10% sodium dodecyl sulfate (SDS) was then added and incubation continued for 30 min at roomtemperature. At this point 200 jig HeLa cell DNA, purified as described previously,7 was added ascarrier to those preparations containing P32-labeled DNA. The mixture was adjusted to 2 XSSC and extracted with an equal volume of redistilled phenol equilibrated with 0.01 M tris bufferpH 7.0. The aqueous phase was re-extracted with fresh phenol and dialyzed against SSC. ForAAV-DNA, papain was re-added after 5 hr and the incubation at 370C continued for 20 hr. Themixture was then adjusted to 0.1 M tris pH 8.6 and incubated for 3 hr at 370C with 100jug trypsin/-ml. Treatment with SDS and subsequent procedures were the same as described for adenovirusDNA extraction. In some instances, SDS-treated incubation mixtures containing P32-labeledviral DNA and carrier HeLa cell DNA were adjusted to a density of 1.70 gm/cm3 with CsCl andcentrifuged at 33,000 rpm for 60 hr in the SW-39 rotor of the Spinco model L centrifuge. Frac-tions were collected, and the acid-insoluble radioactivity and density of each fraction were meas-ured. Fractions containing DNA were pooled and dialyzed against SSC. DNA concentrationswere determined by assuming an optical density at 260 miz of 1.0 for a solution containing 50'ug/ml.

Base composition of viral DNA: Viruses were propagated in the presence of medium containing4 X 10-4 M phosphate and 0.02 me/ml P32-orthophosphate. P32-labeled viral DNA mixed withcarrier HeLa cell DNA (prepared by phenol extraction or preparative CsCl gradients) was digestedto 5'-mononucleotides with pancreatic DNase and snake venom phosphodiesterase.8 After addi-tion of 1 mg carrier 5'-deoxynucleotides, the base composition of the enzymatically hydrolyzed,P32-labeled DNA was calculated from isotope distribution among the electrophoretically separatednucleotides.9

Density of DNA: Buoyant densities of native and alkali-treated viral DNA were determinedin equilibrium density gradients of a CsCl solution (0.001 M sodium EDTA, 0.01 M tris pH 8.6,p = 1.710 gm/cm3). Cl. perfringens DNA, kindly provided by Dr. K. Kohn, was added as amarker. Centrifugations were performed at 250C and 44,770 rpm in the Spinco model E ultra-centrifuge equipped with ultraviolet absorption optics, and photographic negatives were analyzedwith the Spinco Analytrol densitometer. For denaturation, DNA samples in 0.1 X SSC wereexposed to 0.2 N NaOH for 10 min at room temperature and then neutralized.

Thermal melting of DNA: Temperature and optical density measurements of DNA samples in0.1 X SSC and SSC were made in a Beckman spectrophotometer equipped with a Gilford 2000multiple sample absorbance recorder.

Determination of DNA sedimentation coefficients: DNA was sedimented in 1.0 M NaCl, 0.01 Mtris buffer pH 7.6 at 200C and 52,640 rpm in the model E ultracentrifuge. AAV-DNA was sedi-mented at a concentration of 34 ,g/ml, and adenovirus type 2 DNA was sedimented at concentra-tions of 40, 20, and 10 ug/ml.A0 Photographs taken at 4-min intervals were analyzed with theAnalytrol densitometer.Zonal centrifugation of DNA in sucrose gradients: Adenovirus type 2 DNA-H3 and AAV-

DNA-P32 were sedimented in 5-20% sucrose gradients as described by Burgi and Hershey."'Precise 0. 1-ml fractions were collected through a puncture in the bottom of the centrifuge tube bydisplacement with mineral oil added at the top in volumes measured with a microburet (Micro-Metric Instrument Co.).

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Measurement of radioactivity: Acid-insoluble radioactivity was precipitated with 5% trichloro-acetic acid, collected on Millipore filters, dried, and counted in toluene containing Liquifluor(Nuclear-Chicago) in a scintillation counter.

Results.-Purified virus preparations: After three cycles of centrifugation inCsCl density gradients, the AAV band at a density of 1.40 gm/cm3 did not containdetectable E46+ by immunological and electron microscopic examination. SinceE46+ in these preparations could not be similarly freed of AAV, E46+ was producedfrom stocks without detectable AAV. It had a density of 1.35 gm/cm8n. No AAVcontamination of purified E46+, E46-, and adenovirus type 2 preparations wasfound. Purified AAV and E46+ have no common CF antigen and specific neutral-izing antibodies do not cross-neutralize.2

Viral DNA detection and extraction: As shown in Figure 1, thymidine-H3 wasincorporated into AAV and E46+ particles, indicating that they contain DNA.Radioactive peaks at a density of 1.35 gm/cm3 correspond to the adenovirus bandsin both preparations. The second radioactive band at a density of 1.40 gm/cm3in gradient B corresponds to the position of the AAV band. The absence of AAVfrom gradient A was confirmed by CF and electron microscopic examination.DNA was extracted from purified adenovirus in 60-80 per cent yields by a modi-

fication of the method described by Green and Pifia.'2 AAV-DNA could not beefficiently extracted using the same procedure, but with further modification (seeExperimental Procedure) a 60-80 per cent recovery of DNA was also obtained. Todetermine whether purified AAV preparations might be contaminated by adsorbedDNA, P32-labeled virus was incubated in 0.015 M MgCl2, 0.15 M tris pH 6.9 with

400 A

Inative200 ~~~~~~~~~~~~~~~~~~AAV

2 .0 1

DENSITY (gm/ 1.6 71 denatured

I>E200Tt _ AAVI B o a.729

UZi nativeof+peaain.phtgpE46s100 a

I 711wasadded1aeiftdenatured(A)E46whtecbDA169

0'1.50 145 140 1.35

DENSITY ( cn/Clann) 1.691 1.726DENSITYFIG. 1.-Patterns of acid-insoluble

radioactivity in 0.2-ml fractions from FIG. 2.-Densitom-initial CsCl density equilibrium gradients eter tracings fromof E46 + preparations. Thymidine-H3 photographs of DNA(10 junc/ml, sp. act. = 2-3 c/mmole) in CsCl density gra-was added 1 hr after infection. In- dients at equilibrium.cubation and preliminary purification as The marker in eachdescribed in Experimental Procedure. case is Cl. perfringens(A) E46+ inoculum without detectable DNA of density 1.691AAV. (B) E46 + inoculum containing gm/cm3. DNA sam-AAV. pies were 0.5-1.5sg.

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TABLE 1BASE COMPOSITION OF AAV AND ADENOVIRUS DNA

Moles/100 Moles NucleotidesDNA Adenylic Thymidylic Guanylic Cytidylic Guanylic +source acid acid acid acid cytidylic acidAAV 22.9 ± 0.1 22.8 ± 0.1 26.9 ± 0.2 27.3 ± 0.3 54.2 ± 0.2E46+ 24.5 i 0.5 25.5 ± 0.3 25.2 ± 0.9 24.8 ± 0.1 50.0 ± 0.8E46- 25.0 i 0.3 25.9 i 0 24.3 ±4 0.5 24.8 i 0.2 49.1 i 0.3

Results are expressed as mean value ± standard error of the mean. The values are based on fourseparate determinations for E46+ and E46- DNA and six separate determinations for AAV-DNA. Themethod of DNA purification (CsCI density gradient or phenol extraction) had no effect on the resultsof base composition.

60 ,lg pancreatic DNase/ml at 370C for 1 hr. After this treatment 98 per cent ofthe radioactivity remained acid-insoluble.

Properties of Viral DNA.-(a) Base composition: The base composition of AAVand E46+ DNA is given in Table 1. Purine-to-pyrimidine ratios in each case areclose to unity as expected for double-stranded DNA.The 54.2 per cent guanylic plus cytidylic acid (G + C) content of AAV-DNA

differs significantly from the 50.0 per cent G + C content of E46+ DNA. Alsoshown is the base composition of DNA from E46- which lacks the capacity toinduce SV40 tumor antigen. Its base composition is indistinguishable from thatof E46+.

(b) Buoyant density: The buoyant densities of native AAV-DNA and E46+DNA are 1.717 gm/cm3 and 1.711 gm/cm3, respectively (Fig. 2). After exposureto 0.2 N NaOH and neutralization, both DNA's undergo buoyant density increasesexpected for alkali denaturation of helical, double-stranded DNA (Fig. 2).1' TheG + C content predicted from buoyant density values of native DNA14 is 58.2 percent for AAV and 52.0 per cent for E46+.

(c) Thermal melting: Thermal melting profiles of AAV-DNA and E46+ DNAin SSC are shown in Figure 3. AAV-DNA undergoes a sharp transition with a Tmof 92°C, and E46+ DNA a broader transition with a Tm of 90°C. The sharp tran-sition of AAV-DNA suggests relative homogeneity of base distribution within themolecule. In both cases the hyperchromic shiftsare those of helical, double-stranded DNA's. The 1.4 E46*G + C content based on Tm in SSC'5 is 55.4 percent for AAV-DNA and 50.5 per cent for E46+ E AAVDNA. 13_AAV-DNA melted in 0.1 X SSC (Tm 780C) and m

heated to 1010C was fast-cooled in ice water. °There was no decrease in optical density. This 2/finding indicates that the DNA is not cross- 7linked.'6 However, when AAV-DNA was heatedto 1010C in SSC and fast-cooled, there was a re-turn to the initial optical density. The meltingcurve of this fast-cooled DNA was the same asthat for the native material. A rapid and effi- 75 61 85 90 95 100cient reannealing of AAV-DNA after melting in TEMPERATURECSSC could be due to retention of residual ordered ofFIG. 3.-Thermal melting curves

Of AAV-DNA and E46+ DNA, 20sections.'6 Alternatively, reannealing of com- ig/ml each.

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FIG. 4.-Boundary sedimentation of AAIV DNA. Composite of boundary photographs beginning7 min after reaching nominal speed and taken at 8-mmn intervals thereafter.

pletely separated strands may occur rapidly in a uniform population of small DNAmolecules.17

(d) Molecular weight: An S20,,, value of 14.4 was calculated for AAV-DNAfrom boundary sedimentation data (Fig. 4). According to relationships proposedby Eigner and Doty,"8 the sedimentation coefficient corrected to zero concentrationis 15.5, and the estimated molecular weight is 3.5 X 106 daltons.

Molecular weight was also estimated by DNA sedimentation in 5-20 per centsucrose density gradients according to the method of Burgi and Hershey" (Fig. 5).Adenovirus type 2 DNA was used as a reference for determining the molecularweight of AAV-DNA. The adenovirus type 2 DNA has a molecular weight of21 X 106 daltons calculated from its sedimentation coefficient corrected to zeroconcentration."'When the relative distances sedimented by adenovirus and AAV-DNA's are related to the molecular weight of adenovirus DNA, the molecularweight of AAV-DNA is 3.6 X 106 daltons. This figure is in excellent agreementwith that based on boundary sedimentation.

Discussion and Conclusions.-AAV contains DNA which differs from that ofE46+ with respect to base composition, buoyant density, melting profile, and mo-lecular weight. The T. and buoyant density of E46+ DNA and the Tm of AAV-DNA are in good agreement with the chemically determined G + C values. How-ever, the 58.2 per cent G + C content predicted from AAV-DNA buoyant densityis high in comparison with 54.2 per cent as measured directly. Further studieswill be required to evaluate the significance of this difference.

It has been shown that a fraction of the E46+ adenovirus particles contains

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SV40 genetic material,19' 20

while no demonstrable SV40 ,000- 375genetic determinant is found in -Adenovirus 2 DNA-H3the E46- substrain. Since theaverage G + C content of 0 . 250lSV40 DNA (41%)2 is lower .AAVDNA-P32 0

than that of E46 DNA (49- 2550%), the possibility of de- 5,0015-tecting a difference in base .composition between E46+ andE46- DNA was considered. 0, 2'0 30 40

However, the base composi-VOUECLCTD(lHowever ,the base composi- FIG. 5.-Sedimentation of adenovirus type 2 DNA-H3tions of these DNA's were and AAV-DNA-P'2 in a gradient of 5-20% sucrose, 0.1indistinguishable (Table 1). I NaCl, 0.05 M sodium phosphate pH 6.6. Adenovirus

DNA - 0.5 ;Lg and AAV-DNA - 0.25 Atg in 0.1 ml ofFurthermore, native as well as buffered saline were layered onto 4.8 ml of sucrose solu-denatured E46+ DNA formed tion and centrifuged at 32,000 rpm in the SW-39 rotor of

the model L Spinco centrifuge for 6 hr at 40C. Acid-a single band in CsCl density insoluble P32 and H3/0.1-ml fraction are shown.gradients (Fig. 2). Thesefindings suggest that the SV40 genetic material is a small fraction of the total E46+DNA.The DNA's from polyoma, SV40, and rabbit papilloma viruses have molecular

weights21-25 near that calculated for AAV-DNA, and consist in part of circularduplex molecules.21 25 26 When the extracted AAV-DNA was denatured with heator alkali, no component indicative of circular or cross-linked molecules was found.However, the presence of circular DNA with strand scissions cannot be excluded.The molecular weight of 3.6 X 106 daltons is based on the assumption that thisDNA is linear and not degraded.

It should be stressed that although AAV could be purified from E46+ in CsCldensity gradients, the E46+ component could not similarly be freed of AAV. It isevident that adenoviruses purified by density gradient techniques may be con-taminated with AAV.Both the DNA and antigenic component (or components) of this AAV differ

from those of E46+, but a genetic relationship between these viruses cannot beexcluded. Nucleic acid homology experiments will be required to test this possi-bility. Recently, two additional AAV's designated serotypes 2 and 32 have beenpurified from adenovirus preparations and shown to contain DNA.27 Character-ization of these other AAV-DNA's and studies of the genetic relatedness of AAVtypes to each other and to adenoviruses may provide insight into the mechanismby which adenovirus influences AAV replication.

The authors thank Dr. Kurt Kohn for many helpful discussions. The excellent technicalassistance of Mr. Frank Koczot and Mr. David Reigle is gratefully acknowledged.

* These findings were reported in part at the meeting of Federation of American Societies forExperimental Biology, April 1966 [Federation Proc., 25, 684 (1966)].

1 Atchison, R. W., B. Casto, and W. Hammon, Science, 149, 754 (1965).2 Hoggan, M. D., H. Blacklow, and W. P. Rowe, these PROCEEDINGS, 55, 1467 (1966).3 Huebner, R. J., R. M. Chanock, B. Rubin, and M. J. Casey, these PROCEEDINGS, 52, 1333

(1964).

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4 Rowe, W. P., and S. G. Baum, these PROCEEDINGS, 52, 1340 (1964).5 Rose, J. A., P. Reich, and S. Weissman, Virology, 27, 571 (1965).6 Green, M., and M. Pifia, Virology, 20, 199 (1963).7 Marmur, J., J. Mol. Biol., 3, 208 (1961).8 Unpublished method of C. Patch and N. P. Salzman modified from Lehman et al. [Lehman,

I. R., M. Bessman, E. Simms, and A. Kornberg, J. Biol. Chem., 233, 163 (1958)].9 Sebring, E., and N. P. Salzman, Anal. Biochem., 8, 126 (1964).10 This DNA had an S'20,. of 31 by extrapolation of the S20.w values to zero concentration.11 Burgi, E., and A. D. Hershey, Biophys. J., 3, 309 (1963).12 Green, M., and M. Pifia, these PROCEEDINGS, 51, 1251 (1964).13 Vinograd, J., J. Morris, N. Davidson, and W. Dove, these PROCEEDINGS, 49, 12 (1962).14 Schildkraut, C. L., J. Marmur, and P. Doty, J. Mol. Biol., 4, 430 (1963).15 Marmur, J., and P. Doty, J. Mol. Biol., 5, 109 (1962).16 Geiduschek, E. P., J. Mol. Biol., 4, 467 (1962).17 Marmur, J., and P. Doty, J.. Mol. Biol., 3, 585 (1961).18 Eigner, J., and P. Doty, J. Mol. Biol., 12, 549 (1965).19 Rowe, W. P., and S. G. Baum, these PROCEEDINGS, 52, 1340 (1964).20 Rapp, F., J. L. Melnick, J. S. Butel, and T. Kitahara, these PROCEEDINGS, 52, 1348 (1964).21 Crawford, L. V., and P. H. Black, Virology, 24, 388 (1964).22 Weil, R., and J. Vinograd, these PROCEEDINGS, 50, 730 (1963).23 Crawford, L. V., Virology, 22, 149 (1964).24 Mayor, H. D., R. M. Jamison, and L. E. Jordan, Virology, 19, 359 (1963).25 Crawford, L. V., J. Mol. Biol., 8, 489 (1964).26 Dulbecco, R., and M. Vogt, these PROCEEDINGS, 50, 236 (1963).27 Rose, J. A., M. D. Hoggan, and A. J. Shatkin, unpublished results.

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