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Defective-interfering particles of the human parvovirus adeno-associated virus
VIROLOGY94, 162-174 (1979)
Defective-Interfering Particles of the Human Parvovirus Adeno-Associated Virus CATHERINE
A. LAUGHLIN,
MAUREEN W. MYERS, J. CARTER1
DEBRA
L. RISIN,
AND BARRIE Laboratory of Experimental
Pathology, National Institute of Arthritis, Metabolism, and Digestive National Institutes of Health, Bethesda, Maryland 20014
Diseases,
Accepted December 5, 1978
We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV(1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV particles. These infectious AAV particles exhibited autointerference. The light-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious but interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5 S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus. INTRODUCTION
Most parvoviruses produce several classes of particles as assayed by buoyant density equilibrium sedimentation in CsCl [See Rose (19’74) and Siegl (1976) for reviews]. When adeno-associated virus (AAV) was grown in human KB cells in the presence of a helper adenovirus several classes of AAV particles were produced as defined by their buoyant density in CsCl. The predominant classes of AAV particles banded in CsCl at densities of 1.45, 1.41, 1.35, and 1.32 g/cm3 and were referred to as AAV particles AAV(1.45), AAV(1.41), AAV(1.35), and AAV(1.32), respectively (de la Maza and Carter, 1978). The AAV(1.41) or (1.45) particles (i.e., standard AAV) were the only particles ’ To whom reprint requests should be addressed. 004%6822/79/050162-13$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reprodmtion in my form reserved.
162
containing unit length, 14.5 S DNA genomes. The AAV(1.35) and (1.32) particles contained AAV DNA molecules that were about 20% or 3 to 5%, respectively, of genome length. These deleted AAV DNA molecules were enriched for left or right terminal regions of the genome. They appear to constitute a mixed population of molecules including some which can “snap back” to yield crosslinked duplexes and others which annealed to yield normal (noncrosslinked) duplexes. It was also shown (de la Maza and Carter, 1978) that some particles (about 5- 10% of the total) in the standard AAV preparations contained aberrant genomes which were close to unit length but could snap back to form crosslinked duplexes. These molecules were also enriched for terminal regions of the genome. Thus, given density particles
ADENO-ASSOCIATED
VIRUS
DEFECTIVE-INTERFERING
appear to contain given size classes of AAV DNA. While most density classes of aberrant genomes appeared to be represented, the AAV(1.35) particles, containing 10 S DNA genomes, were the most predominant. Also, the 1.32 particles containing very small genomes, were not readily distinguished from completely empty particles banding at a similar density. When the standard AAV species were grown in cells coinfected with adenovirus they exhibited autointerference (Carter et al., 1979). As the multiplicity of infection with standard AAV was increased, the yield of infectious progeny AAV decreased. This appeared to be correlated with a decrease in production of standard AAV DNA genomes and a concomitant increase in production of shorter, aberrant AAV genomes. It was suggested that this autointerference might be due to defective-interfering (DI) particles present in the preparations of standard virus. The small proportion of particles in the standard preparations that contain aberrant, deleted genomes are likely candidates for putative DI particles. However, these aberrant particles cannot be separated readily from standard AAV and therefore we have analyzed the biological properties of the AAV(1.35) and AAV(1.32) particles. We show that the lower density AAV particles are noninfectious but that at least some appear to be DI particles that interfere with AAV(1.41) multiplication. This interference with standard AAV multiplication occurred intracellularly, in part at the level of AAV DNA synthesis. In contrast to AAV(1.41), the AAV(1.35), and AAV(1.32) particles did not inhibit replication of adenovirus. These results suggest that the AAV autointerference phenomenon may be due to AAV DI particles.
PARTICLES
163
three cycles of banding in CsCl equilibrium buoyant density gradients, as discussed further in the text. All AAV preparations used for experiments were heated in the text. All AAV preparations used for experiments were heated to 60” for 15 min to inactivate contaminating adenovirus. KB cells were grown in monolayer cultures in Eagle’s medium containing 5% fetal calf serum. Cell lines are routinely checked by standard procedures for absence of mycoplasma contamination. Cells were grown to 50-70% confluency, then infected with Ad2, usually at a multiplicity of infection (m.o.i.> of 5 plaque forming units (PFU)/cell, and an appropriate multiplicity of AAV. For DNA replication experiments, the KB cells were grown in 75-cm2T-flasks (Falcon Plastics). Virus dilutions were made in phosphate-buffered saline (PBS), pH 7.2, containing 2% dialyzed fetal calf serum. Adsorption of virus was performed in a 2.0-ml volume at room temperature for 1 hr. The inoculum was then removed, 10 ml of Eagle’s medium containing 2% dialyzed fetal calf serum was added, and the infected cultures were incubated at 37”. For experiments using cells on microscope slides adsorption of virus was performed as described below. Isotopic labeling and analysis of intracellular viral DNA. Infected cell cultures (75~cm2T-flasks) were labeled with [methyl3H]thymidine (specific activity 40-60 Ci/ mmol) at a concentration of 25 to 50 $X/ml for 4 hr from 16 to 20 hr after infection. The medium was then removed and the cells lysed by the method of Hirt (1967) including digestion with Pronase as described elsewhere (Carter et al., 1979). The lysed culture was precipitated overnight at 4” with NaCl at a final concentration of 1.1 M then centrifuged at 10,000 rpm for 20 min in the HB-4 rotor of the Sorvall RC-5 centrifuge. The supernatant was dialyzed MATERIALS AND METHODS for 72 hr against three changes of 4 liters Virus and cells. Stocks of adeno-associated of either 0.15 M NaCl, 10 mM Tris (pH S.O>, virus type 2 (AAV-2H) and adenovirus 1 mM EDTA, or 1 x SSC (0.15 M NaCI, type 2 were grown in KB-3 cells in 0.015 M sodium citrate), 1 mJ4 EDTA. suspension culture at 37” (Carter et al., Under these conditions the AAV DNA 1973). AAV particles were purified from usually reannealed, and to ensure this, the cell lysates using the trypsin-deoxycholate dialyzed DNA was incubated at 66” for 1 hr procedure (Rose et al., 1971), followed by prior to velocity sedimentation.
164
LAUGHLIN
Sedimentation analysis of virus particles and DNA. DNA preparations were sedimented in 5-20% sucrose gradients (12.0 ml) containing 1 M NaCI, 10 mM Tris (pH S.O), 1 mJ4 EDTA in the Beckman SW41 rotor for 14-17 hr at 28,000 rpm and 20” in either a Beckman ultracentrifuge or a Sorvall OTD 65 ultracentrifuge. AAV virions stored in CsCl were dialyzed for several hours in a collodion bag against a buffer containing 0.3 2M NaCl, 50 mM Tris-HCI (pH S.O>,1 mM Na,EDTA. The virus (0.2 ml) was then layered on a gradient (11.2 ml) of 15-30% sucrose in 1 M NaCI, 50 mM Tris-HCl (pH S.O>,formed over a shelf (0.8 ml) consisting of CsCl of density 1.834 g/cm3. The gradients were centrifuged in the SW41 rotor in a Beckman ultracentrifuge for 2 hr at 35,000 rpm and 15”. Gradients were collected by bottom puncture. Assay of viral infectivity. Infectious units of AAV were measured using a fluorescent focus assay as described in detail elsewhere (Carter et al., 1979). Briefly, KB cells were grown in a monolayer on microscope slides containing eight chambers (Lab-Tek Products, Naperville, Ill.). The cells were infected with the helper adenovirus at an m.o.i. of 5-10 PFU per cell and an appropriate dilution of the AAV preparation. Cells were fixed at 30 hr after infection and stored at -20” until stained. The number of cell nuclei containing AAV protein was then determined by an indirect immunofluorescent assay using anti-AAV-2 rabbit IgG followed by FITC-conjugated hamster anti-rabbit IgG (Miles Laboratories, Elkhart, Ind.). Adenovirus infectivity was measured using a plaque formation assay on monolayers of KB cells at 37”. Estimation of virus particle numbers and particle to infectivity ratio. AAV &ions purified by banding to equilibrium in CsCl three times were further purified by velocity sedimentation in a sucrose gradient to remove contaminating adenovirus particles. Fractions of the sucrose gradients containing the peak of AAV (see Fig. 1) were pooled and extensively dialyzed against 10 mJ4 Tris-HCl (pH S.O), 1 IM NaCI. The absorbance at 280 nm was then measured. Protein concentration was estimated using the procedure of Lowry
ET AL. a
0.6
0.4
0.2
1
3.0
g 2.0 54 1.0
E 2 a
0.05
0.3
0.2
0.1
10
20
30
FRACTION NUMBER
FIG. 1. Sedimentation of AAV particles in neutral sucrose gradients. Aliquots of the virus bands from the preparation described in Fig. 1 and Table 1 were sedimented as described under Materials and Methods. The gradients were fractionated and the absorbance of each fraction was determined at 260 (0) and at 280 nm (0). The adenovirus particles present in the 1.35 and 1.32 preparations were collected on the CsCl cushion (fractions l-3). (a) AAV(1.41); (b) AAV(1.35); (c) AAV(1.32).
ADENO-ASSOCIATED
VIRUS
DEFECTIVE-INTERFERING
(1951) and serum albumin as a standard. From these data the values for e28,,10/C protein obtained were 61.2, 26.6, and 19.0 for AAV (1.41), (1.35), or (1.32), respectively. These numbers are consistent with those calculated for full and empty particles of minute virus of mice (Tattersall et al., 1976). Particle numbers were then calculated using these extinction coefficients and the known DNA and protein content of AAV particles (1.41 x lo6 and 4.0 x lo”, respectively, see Table 1). The estimation of AAV particle numbers made in this way for AAV(1.41) and AAV(1.35) agreed well with estimations made on virus preparations containing “2P-labeled AAV DNA in which particle numbers were calculated from the amount and specific activity of the radioactive DNA. For AAV(1.32) particles, estimations based on DNA content seriously underestimated the particle number because of the presence of excess empty particles (see text). Ultraviolet irradiation of virus particles. For uv inactivation of virus particles, virus dilutions (1.0 ml) were placed in 6-cm plastic petri dishes and exposed to 4 ~W/mm2 of uv irradiation. This dose of uv for 30 min was sufficient to inactivate infectivity of AAV (1.41) by a factor of at least lo”.
et al.
TABLE
Adsorption
PARTICLES
of virus
165
particles
to cells.
Dilutions of a preparation of AAV(1.41) particles containing 32P-labeled DNA were made in medium without serum and aliquots (0.1 ml or 0.2 ml) were added to 2 x lo” KB cells grown in a monolayer in microscope slide chambers (Lab-Tek). After adsorption of virus for 2 hr at room temperature, the cell sheet was rinsed several times with ice-cold PBS to remove unadsorbed particles. The cell sheet was removed from the chamber with trypsin and mixed with an equal volume of 20% trichloroacetic acid. The 32Pcontent of the resulting precipitate was determined by measuring Cerenkov radiation to determine the amount of 32P-labeled virus that had been bound to the cells. For determination of the effect of AAV( 1.35) or AAV( 1.32) particles on adsorption of AAV(1.41), appropriate amounts were added to the cells together with the 1.41 particles during the 2-hr adsorption period. RESULTS
Purification
and Chayacterixation
of AAV
Particles
The AAV2 virus particles from lysates of KB cells infected with AAV2 and Ad2 1
PROPERTIES OF AAV PARTICLES
Particles” AAV(l.41) AAV(1.35) AAV(1.32)
Sedimentation coefficientb (S) 111 78 66
Concentration’ (particles/ml) 18.7 x 10’3 4.4 x 10’3 12.8 x 10’3
Infectivityd (infectious units/ml)
Particles/ infectious unite
4.4 x 10’”
42 >4.4 x 10’ >12.8 x 107
o The particles were obtained from a lo-liter culture of KB-3 spinner cells (a total of 3.0 x lo9 cells) infected at a multiplicity of 20-40 infectious units/cell with AAV2 and 5- 10 PFUicell of Ad2. * Sedimentation coefficients were determined in 15-30s sucrose gradients at pH 8.0 containing 1 M NaCl using 3H-labeled-MVM (minute virus of mice) obtained from D. Ward as a sedimentation marker (de la Maza et al., in preparation). c The number of particles per milliliter was determined as described under Materials and Methods using the %3 lqOProteinand taking 1.41 x lo6 as the molecular weight of AAV DNA (Koczot et al., 1973; Gerry et al., 1973) and 5.4 x lo6 as the molecular weight of a full AAV particie (Rose et al., 1971). The volume of each particle preparation was 2.0 ml and therefore the total yield of particles was about twofold that shown. d Infectivity was measured as described under Materials and Methods using KB cells. e The ratio of particles to infectious virus was calculated from the data in columns 3 and 4.
166
LAUGHLIN
ET AL.
were purified by equilibrium sedimentation in CsCl to separate the various density classes. Each of the indicated density 1.0 species was individually banded to equilibrium twice more in CsCl gradients. The final purified bands were stored in CsCl at 0.8 4”. There was no loss of AAV titer over $ several months. Aliquots of each band were % 0.6 also sedimented through neutral sucrose B gradients and the uv absorbance profiles 2 of the AAV components were analyzed. 0.4 The properties of these virus preparations are shown in Figs. 1 and 2 and summarized in Table 1. 0.2 As shown in Fig. la, the AAV(1.41) component was a single AAV species which sedimented at 111 S. This component repre240 250 2Lw 270 280 290 300 310 sented infectious, full (standard) AAV WAVELENGTH Inm) particles and had a uv spectrum (Fig. 2) FIG. 2. Absorption spectra for AAV particles. with an absorption maximum at 260 nm and a 260/280 ratio of 1.38. The AAV( 1.32) AAV(1.41), AAV(1.35), or AAV(1.32) particles were component contained a peak of AAV par- purified by CsCl banding and subsequent velocity sedimentation as described in Fig. 1. Absorption ticles which sedimented at approximately was determined in a buffer containing 10mM Tris-HCl 66 S (Fig. lc) and had a uv spectrum (pH 8.0), 1 M NaCl. The absorbance profiles have been (Fig. 2) with an absorption maximum at normalized to maxima of 1 absorbance unit. 280 nm, a shoulder at about 290 nm, and a 2601280ratio of 0.64. The AAV(1.35) comof AAV(1.41) Multiplication by ponent sedimented at approximately 78 S Inhibition AAV(l.35) and AAV(l.3.2) Particles (Fig. lb) and had a uv spectrum (Fig. 2) intermediate between that of the 1.41 and To determine if the AAV( 1.35) and 1.32 species. The AAV(1.35) and AAV(1.32) AAV(1.32) particles behave as DI particles preparations also contained full or partially we first measured their effect upon producempty adenovirus particles, respectively, tion of infectious AAV progeny in KB monowhich sedimented onto the CsCl cushion layer cells infected with AAV(1.41) and a (Fig. 1). The uv spectra of the AAV(1.41) helper adenovirus (Table 2). Addition of and AAV(1.32) particles are very similar to AAV(1.35) particles clearly inhibited prothose reported for the analogous components duction of infectious progeny AAV at low (full and empty particles) of the autonomous particle ratios. The interpretation of the parvovirus, minute virus of mice, MVM results with added AAV(1.32) particles is (Tattersall et al., 1976). As noted by less clear. The apparent enhancement of Tattersall et aE. (1976), the shoulder on the AAV at the low level of added 1.32 AAV spectrum at 290 nm may be due to particles was not consistently observed. tryptophan. However, the only amino acid There was some inhibition by high amounts analysis reported for AAV (Rose et al., of AAV(1.32) particles. This was probably 1971) did not measure tryptophan. As not due to some indirect effect on the cells shown in Table 1, the particle to infectivity since even larger numbers of particles did ratios for the 1.35 and 1.32 species were not inhibit Ad (Tables 3 and 4). That the decreased by at least lo6 in comparison to inhibition by AAV(1.32) particles is much that of the AAV(1.41) particles. In the less efficient than that by (1.35) particles experiments reported here, we have used might be due to the much smaller size of only the 1.41 species as infectious, standard these genomes. The AAV stocks used in AAV and have not analyzed the minor, these experiments (Table 2) were stored AAV(1.45) species which is also infectious. in CsCl and contained heat-inactivated
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
adenovirus. As noted before (Carter et al., 1978), appropriate dilutions of CsCl or heat-inactivated adenovirus when added to infected cells caused no inhibition of standard AAV. Intracellular
Replication of AAV DNA
Intracellular replication of viral DNA in infected cells was analyzed by labeling DNA with [3H]thymidine from 16-20 hr after infection. Viral DNA was then selectively extracted using a Hirt. procedure (Hirt, 1967) and analyzed by sedimentation in neutral sucrose gradients. In cells infected with adenovirus there was synthesis of a large amount of mature 31 S Ad DNA, whereas in mock-infected cells very little labled DNA was extracted into the Hirt supernatant (Fig. 3A). In cells coinfected with adenovirus and AAV(1.41) there was
167
PARTICLES
synthesis of 14.5 S and 10 S AAV DNA species and a significant inhibition of incorporation of label into adenovirus DNA. The 14.5 S AAV DNA contains mainly mature, progeny AAV DNA (i.e., standard or infectious genomes), whereas the 10 S peak contains aberrant, deleted molecules enriched for the terminal regions of AAV DNA (Hauswirth and Berns, 1978; de la Maza and Carter, 1978; Carter et al., 1979). It should be noted that not all the aberrant genomes comprise a discrete 10 S population but that this is the predominant size produced (Carter et al., 19’79). In cells infected with adenovirus and AAV(1.35) particles (Fig. 3B), there was no detectable synthesis of AAV DNA. Similar results were obtained from infections with adenovirus together with AAV(1.32) particles. We have never detected any AAV DNA synthesis resulting from infections
TABLE 2 EFFECT OF ADDED LIGHT-DENSITY AAV PARTICLESON THE YIELD OF INFECTIOUSAAV”
Added light particles* Added particle
Number per cell
Experiment 1 AAV(1.35) AAV(1.35) AAV(1.35) AAV(1.35) AAV(1.35)
None 200 2,000 6,000 20,000
Experiment 2 AAV(1.32) AAV(1.32) AAV(1.32) AAV(1.32) AAV(1.32)
None 700 7,000 21,000 70,000
Ratio of added light particles to AAV(1.41) particles” 1 10 30 100 3.5 35.0 105.0 350.0
Yield of infectious AAVd Infectivity/ml 4.0 4.0 0.45 0.15 0.19
Percentage control
x x x x x
10s 108 10s 108 108
100.0 100.0 11.2 3.8 4.9
1.5 x 5.4 x 1.75 x 1.20 x 0.30 x
109 109 109 109 109
100.0 360.0 117.0 80.0 20.0
a KB monolayer cells (5 x lo5 cells) were infected with Ad2 (5 PFU/cell) and AAV(1.41) particles at 5 infectious units/cell (i.e., 200 particles per cell). 0 AAV(1.35) or AAV(1.32) particles as indicated were added to the cells together with the AAV(1.41) and Ad virus. e The ratio of added AAV light particles to AAV(1.41) particles is calculated using particle numbers determined as described under Materials and Methods. d Infected cultures were grown at 37” for 40 hr and then harvested by freezing and thawing three times. The lysate was clarified by low speed centrifugation and heated at 66” for 15 min to inactivate adenovirus. The AAV infectivity in each cell lysate was then determined using the assay described under Materials and Methods.
LAUGHLIN
168
ET AL,.
TABLE 3 EFFECT OF AAV PARTICLES ON ADENOVIRUS MULTIPLICATIONS
Adenovirus yield for input ofb Time of harvest postinfection (hr)
PFU/ml x lo-’
% Control
250,000 86.000
4 24 24 24
>O.Ol 0.36 0.88 0.76
<3 100 240 210
40
24
ND”
40.000
24
40 86,000
24
Number of added particles per cell
Added AAV particles
-
None None AAV(1.32) AAV(1.35) AAV(1.41) (m.0.i. = 1) AAV(1.41) (m.0.i. = 1000) AAV(1.41) (m.0.i. = 1) plus AAV(1.35)
m.0.i. = 1.0
m.0.i. = 10.0 PFU/ml x lo-’
% Control
0.05 11.9 13.7 14.0
4 100 114 118
-
3.2
21
ND
-
co. 1
ND
-
10.6
86
a KB monolayer cells grown in 3-cm plastic dishes (l-2 x lo6 cells/dish) were infected with Ad2 at a multiplicity of 1 or 10 PFU/cell together with the indicated amount of added AAV particles. Adsorption was allowed to proceed for 90 min at 37” in a volume of 0.3 ml of Eagles medium. The inoculum was then replaced with 3.5 ml of medium containing 2% fetal calf serum and the cultures were harvested at 4 or 24 hr postinfection. b The adenovirus yield was assayed by plaque assay on KB cells after freezing and thawing the lysates three times. c Not done. TABLE 4 EFFECT OF AAV
DNA PARTICLES ON ADENOVIRUS DNA SYNTHESIST
Number of added AAV particles/cell AAV(l.41)
40 (m.0.i. = 1.0) 40 40 40 400 (m.0.i. = 10.0) 400 40 40
AAV(1.35)
AAV(1.32)
-
-
7,000 70,000 700 23,000 70,000 70,000 -
20,000 200,000 20,000 200,000
Ad2 DNA synthesis, percentage of control 100.0 125.0 98.0 103.0 105.0 25.0 21.0 37.0 40.0 10.0 14.0 25.0 63.0
QKB cell monolayers (75% confluent) grown in 75-cm* flasks were infected with Ad2 (2 PFUicell) and the indicated amounts of added AAV particles. Ad2 DNA synthesis was measured by labeling DNA with r3H]thymidine from 16 to 20 hr postinfection. The viral DNA was then selectively extracted using the Hirt procedure described under Materials and Methods. Aliquots of the labeled DNA preparations were centrifuged in neutral sucrose gradients to determine the amount of radioactivity sedimenting as 31-S mature Ad2 DNA duplexes. The control cells infected with Ad2 alone contained 75,730 3H counts/mm of 31 S Ad2 DNA.
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
PARTICLES
169
FIG. 3. Sedimentation of DNA from Hirt supernatant of AAV-infected cells in neutral sucrose gradients. (A) 3H-labeled Hi supernatant DNA from cells infected with Ad2 only at an m.o.i. of 5 PFUl cell (O), Ad2 plus AAV(1.41) at a m.o.i. of 50 infectious units (i.e., 2000 particles) per cell (0), or mockinfected (A), was sedimented in parallel neutral sucrose gradients at 28,000rpm and 20” for 16hr. (B) 3Hlabeled Hirt supernatant DNA from cells infected with Ad2 (m.o.i. = 5.0) plus AAV(1.35) at 2000 narticles aer cell (0) was sedimented in neutral uradients as for panel A. 32P-labeledlinear duplex AAV DNA (Oj was added as a marker.
with AAV(1.35) or AAV(1.32) even using culture was analyzed in neutral sucrose up to 300,000 particles per cell in the gradients. Figure 4A shows the control presence or absence of helper adenovirus. experiment which demonstrates the autoIn contrast, AAV progeny strand DNA interference of AAV(1.41) particles at the level of AAV DNA replication. As the synthesis was detectable by sedimentation assay of Hirt supernatant DNA from cells multiplicity of AAV(1.41) was increased infected with adenovirus together with there was a decrease in 14.5 S DNA AAV(1.41) at a multiplicity of 0.02 (data synthesis and an increase in 10 S DNA not shown). This is equivalent to about synthesis. Over the multiplicity range em1 AAV(1.41) particle per cell. In the absence ployed, the total amount of AAV DNA of helper adenovirus there was no detectable synthesis (i.e., sum of 14.5 S + 10 S DNA) replication of any density class of AAV did not vary greatly. particles including AAV(1.41). Also, AAVThe viral DNA isolated from cells that (1.35) or AAV(1.32) DNA could not be were infected with AAV(1.41) particles at replicated when cells were coinfected with a multiplicity of 1.0 together with increasing either of these species together with helper amounts of AAV(1.35) particles is shown adenovirus and uv-inactivated AAV(1.41) in Fig. 4B. At relatively low levels of particles (see below). AAV(1.35) particles (350 to 3500 particles per cell or a ratio of 1.35:1.41 particles of about 9:l to 9O:l) there was progressive Interference with Replication of Standard inhibition of standard, 14.5 S DNA and a AAV(1.41) by AAV(1.35) or AAV(I 32) proportional increase in 10 S DNA synthesis. In the experiment shown in Figs. 4B At higher levels of AAV(1.35) particles and C, cells were coinfected with Ad2 at a (35,000 per cell or a ratio of 1.35:1.41 of m.o.i. of 5 PFUkell, AAV(1.41) particles 9OO:l) there was a large overall inhibition at a multiplicity of 1.0 (equivalent to about of AAV DNA synthesis including decreased 40 particles per cell), and varying amounts amounts of both 14.5 S and 10 S DNA. of AAV(1.35) or AAV(1.32) particles. In the Importantly, even at this level, the incontrol culture (Fig. 4A), cells were infected hibition of 14.5 S DNA synthesis was with Ad2 and varying amounts of AAV(1.41) proportionally greater than that of 10 S particles. 3H-Labeled, intracellular viral DNA when compared to the control with no DNA from the Hirt supernatant of each added AAV(1.35) particles. Thus, at least
170
ET AL.
LAUGHLIN
at lower levels of added AAV(1.35) particles, the inhibition resembled that seen in the autointerference experiment (compare Figs. 4A and B). These results suggest that 10 S DNA can be made in response to added AAV(1.35) particles but only in the presence of both adenovirus and AAV(1.41) virus as helpers. We conclude from these results that a helper function provided by AAV(1.41) is required for 10 S DNA production. The great inhibition of total AAV DNA synthesis seen when higher amounts of AAV(1.35) particles were added may be interpreted as inhibition of AAV(1.41) to the level at which it could supply insuf6cient helper function for 10 S DNA synthesis (see Discussion). In Fig. 4C are shown similar experiments using added AAV(1.32) particles. As seen in Fig. 4C, there was progressive inhibition of the standard 1.41 virus replication with
increasing amounts of added 1.32 particles through less than that seen with similar numbers of AAV(1.35) particles. Also, there was inhibition rather than stimulation (as in Fig. 4B) of 10 S DNA replication and no detectable stimulation of synthesis of any DNA of the size expected to be in 1.32 particles (about 3-6 S). In this respect the inhibition mediated by AAV( 1.32) particles did not appear to reflect directly that seen in autointerference experiments (Fig. 4A). These experiments suggest that the 1.35 and perhaps the 1.32 particles may be DI particles. However, the experiments did not reliably determine whether any function of the infecting 1.35 or 1.32 genome was necessary for interference or if interference was merely the result of blocking adsorption to the cells of AAV(1.41) particles. These questions were addressed in the experiments described in the following sections.
C
10
33
50
IO
30
50
10
30
50
FRACTION NUMBER
FIG. 4. Analysis of autointerference exhibited by AAV(1.41) particles and interference exhibited by AAV(1.35) and AAV(1.32) particles. Monolayers (75 cm2)of KB cells were infected with adenovirus 2 (3 PFUkell) and various multiplicities of AAV(1.41) and AAV(1.35) or AAV(1.32) particles. The Hirt supernatant DNA (labeled with [3H]thymidine from X-20 hr postinfection) was sedimented in neutral sucrosegradients. (A) Cells wereinfectedwithAAV(l.41) at multiplicities of0.2(A), 1.0 (O), or 10.0(O) infectious units/cell. (B) Cells were infected with AAV(1.41) at an m.o.i. of 1.0 infectious unit (40 particles)/cell, (0) or together with AAV(1.35) at 350 (O), 3500 (A), or 35,000 (A.) particles per cell. (C) Cells were infected with AAV(1.41) at an m.o.i. of 1.0 infectious unit (40 particles)/cell (0) or together with AAV(1.32) at 1000(O), 10,000(A), or 30,000(A) particles per cell. Note: The same gradient profile of DNA from cells infected with AAV(1.41) at an m.o.i. of 1.0 (0) is plotted in all three panels for comparison. This multiplicity represents approximately 40 AAV(1.41) particles per cell.
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
PARTICLES
171
DI-Mediated Inhibition of AAV is not Due to Inhibition of Cell&w Adsorption
a3 2.8 9.2 280 Li 16 53 160 + AAW 351 P.ruc,eE Added Pet Cell ,x,W) AAw1.32, Pamdes mded Per cdl h1w FIG. 5. Effect of AAV DI particles on cellular adsorption of AAV(1.41) and effect of uv irradiation on AAV DI inhibition. KB cells in monolayers were infected with Ad2 at a multiplicity of 5 together with AAV(1.41) and varying amounts of AAV(1.35) or (1.32) particles. (A) Cells were infected with 32P-labeled AAV(1.41) particles at a multiplicity of 200 infectious units (i.e., 8000 particles) per cell together with the indicated amounts of AAV(1.35) particles. The amount of 32P-labeled AAV particles adsorbed was measured as described under Materials and Methods and is expressed as a percentage of the amount bound in the absence of added AAV(1.35) particles [35% of total added 32P-labeled AAV(1.41) particles]. (B) An analogous experiment to that in A except that AAV(1.32) particles were used instead of (1.35) particles. (C) Cells were infected with Ad2 (5 PFlJl cell) and AAV(1.41) (3 infectious units/cell) together with the indicated amounts of AAV(1.35) particles. DNA was labeled at 16 to 20 hr postinfection with [3H]thymidine and extracted and analyzed on neutral sucrose gradients as described under Materials and Methods. The total amount of 14.5 S AAV DNA in gradients was calculated and plotted as shown. The data was corrected for background radioactivity in the analogous regions of a sucrose gradient of DNA from cells infected with Ad2 alone. The amount of 3H-labeled 14.5 S AAV DNA sythesized in the control in the absence of any added AAV(I.35) particles was 2.9 x lo4 countslmin. (@) AAV(1.35) particles not irradiated; (0) AAV(1.35) particles uv irradiated for 20 min. (0) Cells infected with Ad2 (5 PFU/cell), AAV(1.35) particles, and uv-irradiated AAV(1.41) particles (equivalent of 3 infections units/cell). (D) An analogous experiment to (C) except that AAV(1.32) particles were used in place of AAV(1.35) particles. (A) AAV(1.32) particles not irradiated; (V) AAV(1.32) irradiated for 20 min; (A) AAV(1.32) irradiated for 360 min. (C!) Cells infected with Ad2 (5 PFU/cell), AAV(1.32) particles, and uv-irradiated AAV(1.41) particles (equivalent of 3 infections units/cell).
32P-Labeled AAV(1.41) particles were adsorbed to KB monolayer cells at multiplicities of 200 infectious units per cell in the presence of varying amounts of AAV(1.35) or AAV(1.32) particles. It is difficult to perform this experiment with lower multiplicities of 32P-labeled AAV( 1.41) because of limitations on the specific activity of AAV DNA which can be obtained. The cell layer was then washed extensively and the proportion of cell bound 32P-labeled AAV(1.41) was determined (Figs. 5A and B). At the range of concentrations used, the 1.35 and 1.32 particles did not interfere with the proportion of AAV(1.41) particles bound [about 30 to 35% of the total AAV(1.41) particles]. However, as shown in Figs. 5C and D, similar amounts of AAV(1.35) and (1.32) particles showed inhibition of AAV 14.5 S DNA replication. These results suggest that inhibition by AAV DI particles is not mediated at the 1eveI of adsorption to the host cell. Effect of uv Irradiation
on AAV Particles
The effect of uv irradiation on the biological properties of the AAV particles was investigated and the results are summarized in the experiment shown in Figs. 5C and D. In this experiment, AAV particles were uv irradiated for 20 or 360 min. A ZO-min irradiation was sufficient to decrease the infectivity of AAV(I.41) by 105-fold. As shown in Figs. 5C and D, the inhibition of 14.5 S DNA synthesis by the 1.35 or 1.32 particles could be abolished by uv irradiation. This implies; (a) that some function, presumably replication or perhaps transcription, of AAV(1.35) or AAV(1.32) genomes was required in order to obtain interference and (b) that the inhibition was not mediated at the level of adsorption. A ZO-minute exposure to uv light completely destroyed all the DI activity of the 1.35 particles (Fig. 5C) whereas a 360-min exposure was required to inactivate most of the 1.32 particles (Fig. 5D). These observa-
172
LAUGHLIN
tions are qualitatively consistent with the relative target size for the DI activity of the 1.35 and 1.32 genomes expected on the basis of their molecular weights (de la Maza and Carter, 1978). We have not extended these data to provide a quantitative estimate of the target sizes. When KB cells were infected with adenovirus together with the uv-irradiated AAV(1.41) particles, there was no detectable peak of AAV DNA in the Hirt supernatant and the sedimentation profile was indistinguishable from that of an adenovirus-infected control culture (data not shown). Similar results were obtained when the cells were coinfected with adenovirus, uv-irradiated AAV(1.41) and either AAV(1.35) or AAV(1.32) particles (Figs. 5C and D). Again there was no detectable synthesis of AAV progeny strands of either standard or DI virus. This is consistent with the hypothesis suggested above, that replication of AAV DI particles requires a helper function provided by AAV(1.41) particles.
ET AL.
These experiments show that AAV(1.35) and (1.32) particles do not directly inhibit adenovirus in the absense of 1.41 particles and may not do so even in the presence of 1.41 particles. This latter conclusion is more difficult to be certain about because we cannot readily resolve two effects: (a) a direct effect of adenovirus inhibition mediated by replicating AAV(1.35) or (1.32) and (b) an indirect effect of inhibition of AAV(1.41) by AAV(1.35) or (1.32) which may lead in turn to a release of adenovirus inhibition mediated by AAV(1.41). DISCUSSION
The experiments reported here clearly establish the presence of defective-interfering (DI) particles of adeno-associated virus. We have shown that AAV(1.35) and perhaps AAV(1.32) particles, when added to KB cells together with both AAV(1.41) and Ad2 particles, inhibit production of both standard (infectious) AAV progeny and of standard 14.5 S AAV DNA molecules. Several observations indicate that this Effect of AAV Particles on Adenovirus inhibition is mediated by an intracellular Multiplication function of the AAV(1.35) or (1.32) DNA As shown in Table 3, neither AAV(1.35) genome rather than by the viral protein or nor AAV(1.32) particles showed any inhibi- at the level of adsorption of virus. The tion of the yield of infectious adenovirus at inhibitory activity was inactivated by uv an adenovirus multiplicity of either 10 or 1. irradiation. There was a qualitative corThe control experiment (Table 3) showed that relation between the uv dose required for the inhibition of adenovirus by AAV(1.41) inactivation of the inhibition and the size did occur as reported previously (Carter of the inhibitory genome. Inhibition required et al., 1979). a larger number of AAV(1.32) particles than The failure of 1.35 or 1.32 particles to AAV(1.35) particles which may reflect an block production of infectious adenovirus excess of truly empty AAV particles in the particles may be accounted for by the 1.32 population or the small size of the failure to block adenovirus DNA replication AAV(1.32) DNA genomes. The AAV parti(Table 4). In the control experiments, cles containing the largest aberrant genomes AAV(1.41) particles inhibited incorporation cannot be separated readily from AAV(1.41) of isotopic label into adenovirus DNA particles (de la Maza and Carter, 19’78)but (Table 4) and the AAV(1.35) and AAV(1.32) we conclude by analogy that these aberrant preparations inhibited labeling of AAV- virions may also be DI particles. Thus, the (1.41) 14.5 S DNA (not shown). When autointerference exhibited by AAV(1.41) AAV(1.35) or (1.32) particles were added particle preparations (Carter et al., 1979; to cells, together with adenovirus and see also Fig. 5A) is postulated to be AAV(1.41) particles, there was no addi- mediated by DI particles. The AAV DI particles appear to satisfy tional inhibition, or in some cases even less inhibition, of adenovirus multiplica- all the criteria of DI particles listed by tion over that achieved by the AAV(1.41) Huang and Baltimore (1970, 1977). AAV particles alone (Tables 3 and 4). DI particles have predominantly lighter
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
PARTICLES
173
densities in CsCl than standard AAV, for progeny strand DNA production of contain aberrant, deleted genomes (de la standard AAV. Maza and Carter 1978), inhibit replication The above discussion assumes that the of standard genomes intracellularly, and infecting AAV DI genome is in fact repliappear to require a helper function supplied cated. The uv inactivation experiments and by standard AAV (see below). The DI enhancement of DI DNA production by genomes accumulate preferentially at high added DI particles support this assumption multiplicity of infection (Fig. 4A), and DI but do not constitute rigorous proof. An particles accumulate rapidly during high alternative explanation of the above data is multiplicity undiluted passage (M. Myers, that DI genomes might be produced directly, C. A. Laughlin, D. L. Risin, unpublished and only, from standard genome templates experiments). by an aberrant replication. Added exogenous The mechanism of inhibition by AAV DI DI genomes might somehow enhance this particles is not clear. However, the present process by a mechanism which does not results argue that replication of the AAV DI involve use of the added DI genome as a genome is required. The AAV DI genomes replication template. Experiments designed like other viral DI genomes (Huang and to obtain direct proof of replication of added Baltimore, 1977) are enriched for the region DI genomes are currently in progress. We do not know if all or only some of containing the replication origin (Hauswirth and Berns, 1978; de la Maza and Carter, the aberrant genomes in light-density AAV 1978) and thus should be able to replicate. particles are DI molecules. For instance, The results presented here suggest that the genomes in 1.35 g/cm3 particles appear production of AAV DI genomes requires to be topologically heterogeneous and only some are snap-back molecules, whereas a helper factor supplied by standard AAV. Thus, AAV DI genome production is those from the 1.41 or 1.32 particles appear detected only in cells jointly infected with topologically more homogeneous and are DI genomes [or a high multiplicity of AAV- nearly all snap-back molecules (de la Maza (1.41) particles], AAV(1.41>, and Ad particles. and Carter, 1978). More interestingly, some DI genome replication was not detected in aberrant genomes contain either left- or cells infected only by DI particles or in the right-hand terminal regions of the AAV presence of Ad2, or Ad2 together with genome (de la Maza and Carter, 1978) and uv-inactivated AAV(1.41). Also, AAV DI we do not know whether both classes are genomes inhibit AAV replication but do not DI particles. For any DI inhibition which appear to inhibit adenovirus replication. occurred at the level of transcription it is The simplest interpretation of these data possible that only the left-hand end molecules is that in addition to the adenovirus would be inhibitory since this is the probable helper factors for AAV, the production or location of the AAV promoter (Carter replication of progeny AAV DI particles et al., 1976; Jay et al., 1978). requires an AAV-specified helper function. This function must be either one or more of ACKNOWLEDGMENTS the AAV capsid polypeptides or the two We thank R. Friedman for critical review and Billie additional polypeptides generated during posttranslational cleavage of these proteins Healy for expert preparation of the manuscript. since these are the only known products of translation of AAV mRNA (Buller and REFERENCES Rose, 1978). The AAV DI genomes should not produce these proteins because they BULLER, R. M. L., and ROSE,J. A. (1978). Characterization of adeno-associated virus polypeptides syncontain deletions in the codigenic regions thesized in viva and in vitro. In “Replication of of the DNA (Carter et aZ., 1976; de la Maza Mammalian Parvoviruses” (D. C. Ward and P. and Carter, 1978). On the basis of these Tattersall, eds.) pp. 399-410. Cold Spring Harbor. results, we also argue that it is likely Laboratory, Cold Spring Harbor, N. Y. that an AAV capsid polypeptide is required CARTER, B. J., FIFE, K. H., DE LA MAZA, L. M.,
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and BERNS, K. I. (19’76). Genome localization of adeno-associatedvirus RNA. J. Viral. 19,1044- 1053. CARTER, B. J., KOCZOT,F. J., GARRISON,J., ROSE, J. A., and DOLIN, R. (1973). Separate helper functions provided by adenovirus for adenovirusassociated virus multiplication. Nature New Biol. 244, 71-73. CARTER, B. J., LAUGHLIN, C. A., DE LA MAZA, L. M., and MYERS, M. (1979). Adeno-associated virus auto-interference. Virology 92, 449-462. DE LA MAZA,.L. M., and CARTER, B. J. (1978):DNA structure of incomplete adeno-associated virus particles. In “Replication of Mammalian Parvoviruses” (D. C. Ward and P. Tattersall, eds.), pp. 193-204. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. GERRY, H. W., KELLY, T. J., JR., and BERNS, K. I. (1973). Arrangement of nucleotide sequences in adeno-associated virus DNA J. Mol. Biol. 79, 207-225. HAUSWIRTH, W. W., and BERNS, K. I. (1978). Initiation and termination of adeno-associated virus DNA replication. In “Replication of Mammalian Parvoviruses” (D. C. Ward and P. Tattersall, eds.), pp. 257-267. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. HUANG, A. S., and BALTIMORE, D. (1970). Defective viral particles and viral disease processes. Nature (London)
226, 325-327.
HUANG, A. S., and BALTIMORE, D. (1977). Defective interfering animal viruses. In “Comprehensive
ET AL.
Virology” (H. Frankel-Conrat and R. R. Wagner, eds.), Vol. 10, pp 73-116. Plenum, New York. JAY, F. T., LAUGHLIN, C. A., DE LA MAZA, L. M., CARTER,B. J., and COOK, W. (1978). Adenoassociated virus RNA synthesis in wivo and in vitro. In “Replication of Mammalian Parvoviruses” (D. Ward and P. Tattersall, eds.) pp. 385-379. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. KOCZOT,F. J., CARTER, B. J., GARON, C. F., and ROSE,J. A. (1973). Self-complementarity of terminal sequences within plus or minus strands of adenovirus-associated virus DNA. Proc. Nat. Acad. Sci. USA 70, 215-219. LOWRY, 0. H., ROSEBOROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. ROSE,J. A. (1974). Parvovirus reproduction. In “Comprehensive Virology” (H. Frankel-Conrat and R. R. Wagner, eds.), Vol. 3, pp. 1-61. Plenum, New York. ROSE, J. A., MAIZEL, J. V. JR., INMAN, J. K., and SHATKIN, A. J. (1971). Structural proteins of adenovirus-associated virus. J. Viral. 8, 766-770. SIEGL, G. (1976). “The Parvoviruses.” SpringerVerlag, Wien/New York. STRAUSS, S. W., SEBRING, E. D., and ROSE, J. A. (1976). Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc. Nat. Aead. Sci. USA 73, 742-746. TATTERSALL, P., CAWTE, P. J., SHATKIN,A. J., and WARD, D. C. (1976). Three structural polypeptides coded for by minute virus of mice, a parvovirus. J. Viral.
20, 273-289.
Defective-Interfering Particles of the Human Parvovirus Adeno-Associated Virus CATHERINE
A. LAUGHLIN,
MAUREEN W. MYERS, J. CARTER1
DEBRA
L. RISIN,
AND BARRIE Laboratory of Experimental
Pathology, National Institute of Arthritis, Metabolism, and Digestive National Institutes of Health, Bethesda, Maryland 20014
Diseases,
Accepted December 5, 1978
We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV(1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV particles. These infectious AAV particles exhibited autointerference. The light-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious but interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5 S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus. INTRODUCTION
Most parvoviruses produce several classes of particles as assayed by buoyant density equilibrium sedimentation in CsCl [See Rose (19’74) and Siegl (1976) for reviews]. When adeno-associated virus (AAV) was grown in human KB cells in the presence of a helper adenovirus several classes of AAV particles were produced as defined by their buoyant density in CsCl. The predominant classes of AAV particles banded in CsCl at densities of 1.45, 1.41, 1.35, and 1.32 g/cm3 and were referred to as AAV particles AAV(1.45), AAV(1.41), AAV(1.35), and AAV(1.32), respectively (de la Maza and Carter, 1978). The AAV(1.41) or (1.45) particles (i.e., standard AAV) were the only particles ’ To whom reprint requests should be addressed. 004%6822/79/050162-13$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reprodmtion in my form reserved.
162
containing unit length, 14.5 S DNA genomes. The AAV(1.35) and (1.32) particles contained AAV DNA molecules that were about 20% or 3 to 5%, respectively, of genome length. These deleted AAV DNA molecules were enriched for left or right terminal regions of the genome. They appear to constitute a mixed population of molecules including some which can “snap back” to yield crosslinked duplexes and others which annealed to yield normal (noncrosslinked) duplexes. It was also shown (de la Maza and Carter, 1978) that some particles (about 5- 10% of the total) in the standard AAV preparations contained aberrant genomes which were close to unit length but could snap back to form crosslinked duplexes. These molecules were also enriched for terminal regions of the genome. Thus, given density particles
ADENO-ASSOCIATED
VIRUS
DEFECTIVE-INTERFERING
appear to contain given size classes of AAV DNA. While most density classes of aberrant genomes appeared to be represented, the AAV(1.35) particles, containing 10 S DNA genomes, were the most predominant. Also, the 1.32 particles containing very small genomes, were not readily distinguished from completely empty particles banding at a similar density. When the standard AAV species were grown in cells coinfected with adenovirus they exhibited autointerference (Carter et al., 1979). As the multiplicity of infection with standard AAV was increased, the yield of infectious progeny AAV decreased. This appeared to be correlated with a decrease in production of standard AAV DNA genomes and a concomitant increase in production of shorter, aberrant AAV genomes. It was suggested that this autointerference might be due to defective-interfering (DI) particles present in the preparations of standard virus. The small proportion of particles in the standard preparations that contain aberrant, deleted genomes are likely candidates for putative DI particles. However, these aberrant particles cannot be separated readily from standard AAV and therefore we have analyzed the biological properties of the AAV(1.35) and AAV(1.32) particles. We show that the lower density AAV particles are noninfectious but that at least some appear to be DI particles that interfere with AAV(1.41) multiplication. This interference with standard AAV multiplication occurred intracellularly, in part at the level of AAV DNA synthesis. In contrast to AAV(1.41), the AAV(1.35), and AAV(1.32) particles did not inhibit replication of adenovirus. These results suggest that the AAV autointerference phenomenon may be due to AAV DI particles.
PARTICLES
163
three cycles of banding in CsCl equilibrium buoyant density gradients, as discussed further in the text. All AAV preparations used for experiments were heated in the text. All AAV preparations used for experiments were heated to 60” for 15 min to inactivate contaminating adenovirus. KB cells were grown in monolayer cultures in Eagle’s medium containing 5% fetal calf serum. Cell lines are routinely checked by standard procedures for absence of mycoplasma contamination. Cells were grown to 50-70% confluency, then infected with Ad2, usually at a multiplicity of infection (m.o.i.> of 5 plaque forming units (PFU)/cell, and an appropriate multiplicity of AAV. For DNA replication experiments, the KB cells were grown in 75-cm2T-flasks (Falcon Plastics). Virus dilutions were made in phosphate-buffered saline (PBS), pH 7.2, containing 2% dialyzed fetal calf serum. Adsorption of virus was performed in a 2.0-ml volume at room temperature for 1 hr. The inoculum was then removed, 10 ml of Eagle’s medium containing 2% dialyzed fetal calf serum was added, and the infected cultures were incubated at 37”. For experiments using cells on microscope slides adsorption of virus was performed as described below. Isotopic labeling and analysis of intracellular viral DNA. Infected cell cultures (75~cm2T-flasks) were labeled with [methyl3H]thymidine (specific activity 40-60 Ci/ mmol) at a concentration of 25 to 50 $X/ml for 4 hr from 16 to 20 hr after infection. The medium was then removed and the cells lysed by the method of Hirt (1967) including digestion with Pronase as described elsewhere (Carter et al., 1979). The lysed culture was precipitated overnight at 4” with NaCl at a final concentration of 1.1 M then centrifuged at 10,000 rpm for 20 min in the HB-4 rotor of the Sorvall RC-5 centrifuge. The supernatant was dialyzed MATERIALS AND METHODS for 72 hr against three changes of 4 liters Virus and cells. Stocks of adeno-associated of either 0.15 M NaCl, 10 mM Tris (pH S.O>, virus type 2 (AAV-2H) and adenovirus 1 mM EDTA, or 1 x SSC (0.15 M NaCI, type 2 were grown in KB-3 cells in 0.015 M sodium citrate), 1 mJ4 EDTA. suspension culture at 37” (Carter et al., Under these conditions the AAV DNA 1973). AAV particles were purified from usually reannealed, and to ensure this, the cell lysates using the trypsin-deoxycholate dialyzed DNA was incubated at 66” for 1 hr procedure (Rose et al., 1971), followed by prior to velocity sedimentation.
164
LAUGHLIN
Sedimentation analysis of virus particles and DNA. DNA preparations were sedimented in 5-20% sucrose gradients (12.0 ml) containing 1 M NaCI, 10 mM Tris (pH S.O), 1 mJ4 EDTA in the Beckman SW41 rotor for 14-17 hr at 28,000 rpm and 20” in either a Beckman ultracentrifuge or a Sorvall OTD 65 ultracentrifuge. AAV virions stored in CsCl were dialyzed for several hours in a collodion bag against a buffer containing 0.3 2M NaCl, 50 mM Tris-HCI (pH S.O>,1 mM Na,EDTA. The virus (0.2 ml) was then layered on a gradient (11.2 ml) of 15-30% sucrose in 1 M NaCI, 50 mM Tris-HCl (pH S.O>,formed over a shelf (0.8 ml) consisting of CsCl of density 1.834 g/cm3. The gradients were centrifuged in the SW41 rotor in a Beckman ultracentrifuge for 2 hr at 35,000 rpm and 15”. Gradients were collected by bottom puncture. Assay of viral infectivity. Infectious units of AAV were measured using a fluorescent focus assay as described in detail elsewhere (Carter et al., 1979). Briefly, KB cells were grown in a monolayer on microscope slides containing eight chambers (Lab-Tek Products, Naperville, Ill.). The cells were infected with the helper adenovirus at an m.o.i. of 5-10 PFU per cell and an appropriate dilution of the AAV preparation. Cells were fixed at 30 hr after infection and stored at -20” until stained. The number of cell nuclei containing AAV protein was then determined by an indirect immunofluorescent assay using anti-AAV-2 rabbit IgG followed by FITC-conjugated hamster anti-rabbit IgG (Miles Laboratories, Elkhart, Ind.). Adenovirus infectivity was measured using a plaque formation assay on monolayers of KB cells at 37”. Estimation of virus particle numbers and particle to infectivity ratio. AAV &ions purified by banding to equilibrium in CsCl three times were further purified by velocity sedimentation in a sucrose gradient to remove contaminating adenovirus particles. Fractions of the sucrose gradients containing the peak of AAV (see Fig. 1) were pooled and extensively dialyzed against 10 mJ4 Tris-HCl (pH S.O), 1 IM NaCI. The absorbance at 280 nm was then measured. Protein concentration was estimated using the procedure of Lowry
ET AL. a
0.6
0.4
0.2
1
3.0
g 2.0 54 1.0
E 2 a
0.05
0.3
0.2
0.1
10
20
30
FRACTION NUMBER
FIG. 1. Sedimentation of AAV particles in neutral sucrose gradients. Aliquots of the virus bands from the preparation described in Fig. 1 and Table 1 were sedimented as described under Materials and Methods. The gradients were fractionated and the absorbance of each fraction was determined at 260 (0) and at 280 nm (0). The adenovirus particles present in the 1.35 and 1.32 preparations were collected on the CsCl cushion (fractions l-3). (a) AAV(1.41); (b) AAV(1.35); (c) AAV(1.32).
ADENO-ASSOCIATED
VIRUS
DEFECTIVE-INTERFERING
(1951) and serum albumin as a standard. From these data the values for e28,,10/C protein obtained were 61.2, 26.6, and 19.0 for AAV (1.41), (1.35), or (1.32), respectively. These numbers are consistent with those calculated for full and empty particles of minute virus of mice (Tattersall et al., 1976). Particle numbers were then calculated using these extinction coefficients and the known DNA and protein content of AAV particles (1.41 x lo6 and 4.0 x lo”, respectively, see Table 1). The estimation of AAV particle numbers made in this way for AAV(1.41) and AAV(1.35) agreed well with estimations made on virus preparations containing “2P-labeled AAV DNA in which particle numbers were calculated from the amount and specific activity of the radioactive DNA. For AAV(1.32) particles, estimations based on DNA content seriously underestimated the particle number because of the presence of excess empty particles (see text). Ultraviolet irradiation of virus particles. For uv inactivation of virus particles, virus dilutions (1.0 ml) were placed in 6-cm plastic petri dishes and exposed to 4 ~W/mm2 of uv irradiation. This dose of uv for 30 min was sufficient to inactivate infectivity of AAV (1.41) by a factor of at least lo”.
et al.
TABLE
Adsorption
PARTICLES
of virus
165
particles
to cells.
Dilutions of a preparation of AAV(1.41) particles containing 32P-labeled DNA were made in medium without serum and aliquots (0.1 ml or 0.2 ml) were added to 2 x lo” KB cells grown in a monolayer in microscope slide chambers (Lab-Tek). After adsorption of virus for 2 hr at room temperature, the cell sheet was rinsed several times with ice-cold PBS to remove unadsorbed particles. The cell sheet was removed from the chamber with trypsin and mixed with an equal volume of 20% trichloroacetic acid. The 32Pcontent of the resulting precipitate was determined by measuring Cerenkov radiation to determine the amount of 32P-labeled virus that had been bound to the cells. For determination of the effect of AAV( 1.35) or AAV( 1.32) particles on adsorption of AAV(1.41), appropriate amounts were added to the cells together with the 1.41 particles during the 2-hr adsorption period. RESULTS
Purification
and Chayacterixation
of AAV
Particles
The AAV2 virus particles from lysates of KB cells infected with AAV2 and Ad2 1
PROPERTIES OF AAV PARTICLES
Particles” AAV(l.41) AAV(1.35) AAV(1.32)
Sedimentation coefficientb (S) 111 78 66
Concentration’ (particles/ml) 18.7 x 10’3 4.4 x 10’3 12.8 x 10’3
Infectivityd (infectious units/ml)
Particles/ infectious unite
4.4 x 10’”
42 >4.4 x 10’ >12.8 x 107
o The particles were obtained from a lo-liter culture of KB-3 spinner cells (a total of 3.0 x lo9 cells) infected at a multiplicity of 20-40 infectious units/cell with AAV2 and 5- 10 PFUicell of Ad2. * Sedimentation coefficients were determined in 15-30s sucrose gradients at pH 8.0 containing 1 M NaCl using 3H-labeled-MVM (minute virus of mice) obtained from D. Ward as a sedimentation marker (de la Maza et al., in preparation). c The number of particles per milliliter was determined as described under Materials and Methods using the %3 lqOProteinand taking 1.41 x lo6 as the molecular weight of AAV DNA (Koczot et al., 1973; Gerry et al., 1973) and 5.4 x lo6 as the molecular weight of a full AAV particie (Rose et al., 1971). The volume of each particle preparation was 2.0 ml and therefore the total yield of particles was about twofold that shown. d Infectivity was measured as described under Materials and Methods using KB cells. e The ratio of particles to infectious virus was calculated from the data in columns 3 and 4.
166
LAUGHLIN
ET AL.
were purified by equilibrium sedimentation in CsCl to separate the various density classes. Each of the indicated density 1.0 species was individually banded to equilibrium twice more in CsCl gradients. The final purified bands were stored in CsCl at 0.8 4”. There was no loss of AAV titer over $ several months. Aliquots of each band were % 0.6 also sedimented through neutral sucrose B gradients and the uv absorbance profiles 2 of the AAV components were analyzed. 0.4 The properties of these virus preparations are shown in Figs. 1 and 2 and summarized in Table 1. 0.2 As shown in Fig. la, the AAV(1.41) component was a single AAV species which sedimented at 111 S. This component repre240 250 2Lw 270 280 290 300 310 sented infectious, full (standard) AAV WAVELENGTH Inm) particles and had a uv spectrum (Fig. 2) FIG. 2. Absorption spectra for AAV particles. with an absorption maximum at 260 nm and a 260/280 ratio of 1.38. The AAV( 1.32) AAV(1.41), AAV(1.35), or AAV(1.32) particles were component contained a peak of AAV par- purified by CsCl banding and subsequent velocity sedimentation as described in Fig. 1. Absorption ticles which sedimented at approximately was determined in a buffer containing 10mM Tris-HCl 66 S (Fig. lc) and had a uv spectrum (pH 8.0), 1 M NaCl. The absorbance profiles have been (Fig. 2) with an absorption maximum at normalized to maxima of 1 absorbance unit. 280 nm, a shoulder at about 290 nm, and a 2601280ratio of 0.64. The AAV(1.35) comof AAV(1.41) Multiplication by ponent sedimented at approximately 78 S Inhibition AAV(l.35) and AAV(l.3.2) Particles (Fig. lb) and had a uv spectrum (Fig. 2) intermediate between that of the 1.41 and To determine if the AAV( 1.35) and 1.32 species. The AAV(1.35) and AAV(1.32) AAV(1.32) particles behave as DI particles preparations also contained full or partially we first measured their effect upon producempty adenovirus particles, respectively, tion of infectious AAV progeny in KB monowhich sedimented onto the CsCl cushion layer cells infected with AAV(1.41) and a (Fig. 1). The uv spectra of the AAV(1.41) helper adenovirus (Table 2). Addition of and AAV(1.32) particles are very similar to AAV(1.35) particles clearly inhibited prothose reported for the analogous components duction of infectious progeny AAV at low (full and empty particles) of the autonomous particle ratios. The interpretation of the parvovirus, minute virus of mice, MVM results with added AAV(1.32) particles is (Tattersall et al., 1976). As noted by less clear. The apparent enhancement of Tattersall et aE. (1976), the shoulder on the AAV at the low level of added 1.32 AAV spectrum at 290 nm may be due to particles was not consistently observed. tryptophan. However, the only amino acid There was some inhibition by high amounts analysis reported for AAV (Rose et al., of AAV(1.32) particles. This was probably 1971) did not measure tryptophan. As not due to some indirect effect on the cells shown in Table 1, the particle to infectivity since even larger numbers of particles did ratios for the 1.35 and 1.32 species were not inhibit Ad (Tables 3 and 4). That the decreased by at least lo6 in comparison to inhibition by AAV(1.32) particles is much that of the AAV(1.41) particles. In the less efficient than that by (1.35) particles experiments reported here, we have used might be due to the much smaller size of only the 1.41 species as infectious, standard these genomes. The AAV stocks used in AAV and have not analyzed the minor, these experiments (Table 2) were stored AAV(1.45) species which is also infectious. in CsCl and contained heat-inactivated
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
adenovirus. As noted before (Carter et al., 1978), appropriate dilutions of CsCl or heat-inactivated adenovirus when added to infected cells caused no inhibition of standard AAV. Intracellular
Replication of AAV DNA
Intracellular replication of viral DNA in infected cells was analyzed by labeling DNA with [3H]thymidine from 16-20 hr after infection. Viral DNA was then selectively extracted using a Hirt. procedure (Hirt, 1967) and analyzed by sedimentation in neutral sucrose gradients. In cells infected with adenovirus there was synthesis of a large amount of mature 31 S Ad DNA, whereas in mock-infected cells very little labled DNA was extracted into the Hirt supernatant (Fig. 3A). In cells coinfected with adenovirus and AAV(1.41) there was
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synthesis of 14.5 S and 10 S AAV DNA species and a significant inhibition of incorporation of label into adenovirus DNA. The 14.5 S AAV DNA contains mainly mature, progeny AAV DNA (i.e., standard or infectious genomes), whereas the 10 S peak contains aberrant, deleted molecules enriched for the terminal regions of AAV DNA (Hauswirth and Berns, 1978; de la Maza and Carter, 1978; Carter et al., 1979). It should be noted that not all the aberrant genomes comprise a discrete 10 S population but that this is the predominant size produced (Carter et al., 19’79). In cells infected with adenovirus and AAV(1.35) particles (Fig. 3B), there was no detectable synthesis of AAV DNA. Similar results were obtained from infections with adenovirus together with AAV(1.32) particles. We have never detected any AAV DNA synthesis resulting from infections
TABLE 2 EFFECT OF ADDED LIGHT-DENSITY AAV PARTICLESON THE YIELD OF INFECTIOUSAAV”
Added light particles* Added particle
Number per cell
Experiment 1 AAV(1.35) AAV(1.35) AAV(1.35) AAV(1.35) AAV(1.35)
None 200 2,000 6,000 20,000
Experiment 2 AAV(1.32) AAV(1.32) AAV(1.32) AAV(1.32) AAV(1.32)
None 700 7,000 21,000 70,000
Ratio of added light particles to AAV(1.41) particles” 1 10 30 100 3.5 35.0 105.0 350.0
Yield of infectious AAVd Infectivity/ml 4.0 4.0 0.45 0.15 0.19
Percentage control
x x x x x
10s 108 10s 108 108
100.0 100.0 11.2 3.8 4.9
1.5 x 5.4 x 1.75 x 1.20 x 0.30 x
109 109 109 109 109
100.0 360.0 117.0 80.0 20.0
a KB monolayer cells (5 x lo5 cells) were infected with Ad2 (5 PFU/cell) and AAV(1.41) particles at 5 infectious units/cell (i.e., 200 particles per cell). 0 AAV(1.35) or AAV(1.32) particles as indicated were added to the cells together with the AAV(1.41) and Ad virus. e The ratio of added AAV light particles to AAV(1.41) particles is calculated using particle numbers determined as described under Materials and Methods. d Infected cultures were grown at 37” for 40 hr and then harvested by freezing and thawing three times. The lysate was clarified by low speed centrifugation and heated at 66” for 15 min to inactivate adenovirus. The AAV infectivity in each cell lysate was then determined using the assay described under Materials and Methods.
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168
ET AL,.
TABLE 3 EFFECT OF AAV PARTICLES ON ADENOVIRUS MULTIPLICATIONS
Adenovirus yield for input ofb Time of harvest postinfection (hr)
PFU/ml x lo-’
% Control
250,000 86.000
4 24 24 24
>O.Ol 0.36 0.88 0.76
<3 100 240 210
40
24
ND”
40.000
24
40 86,000
24
Number of added particles per cell
Added AAV particles
-
None None AAV(1.32) AAV(1.35) AAV(1.41) (m.0.i. = 1) AAV(1.41) (m.0.i. = 1000) AAV(1.41) (m.0.i. = 1) plus AAV(1.35)
m.0.i. = 1.0
m.0.i. = 10.0 PFU/ml x lo-’
% Control
0.05 11.9 13.7 14.0
4 100 114 118
-
3.2
21
ND
-
co. 1
ND
-
10.6
86
a KB monolayer cells grown in 3-cm plastic dishes (l-2 x lo6 cells/dish) were infected with Ad2 at a multiplicity of 1 or 10 PFU/cell together with the indicated amount of added AAV particles. Adsorption was allowed to proceed for 90 min at 37” in a volume of 0.3 ml of Eagles medium. The inoculum was then replaced with 3.5 ml of medium containing 2% fetal calf serum and the cultures were harvested at 4 or 24 hr postinfection. b The adenovirus yield was assayed by plaque assay on KB cells after freezing and thawing the lysates three times. c Not done. TABLE 4 EFFECT OF AAV
DNA PARTICLES ON ADENOVIRUS DNA SYNTHESIST
Number of added AAV particles/cell AAV(l.41)
40 (m.0.i. = 1.0) 40 40 40 400 (m.0.i. = 10.0) 400 40 40
AAV(1.35)
AAV(1.32)
-
-
7,000 70,000 700 23,000 70,000 70,000 -
20,000 200,000 20,000 200,000
Ad2 DNA synthesis, percentage of control 100.0 125.0 98.0 103.0 105.0 25.0 21.0 37.0 40.0 10.0 14.0 25.0 63.0
QKB cell monolayers (75% confluent) grown in 75-cm* flasks were infected with Ad2 (2 PFUicell) and the indicated amounts of added AAV particles. Ad2 DNA synthesis was measured by labeling DNA with r3H]thymidine from 16 to 20 hr postinfection. The viral DNA was then selectively extracted using the Hirt procedure described under Materials and Methods. Aliquots of the labeled DNA preparations were centrifuged in neutral sucrose gradients to determine the amount of radioactivity sedimenting as 31-S mature Ad2 DNA duplexes. The control cells infected with Ad2 alone contained 75,730 3H counts/mm of 31 S Ad2 DNA.
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
PARTICLES
169
FIG. 3. Sedimentation of DNA from Hirt supernatant of AAV-infected cells in neutral sucrose gradients. (A) 3H-labeled Hi supernatant DNA from cells infected with Ad2 only at an m.o.i. of 5 PFUl cell (O), Ad2 plus AAV(1.41) at a m.o.i. of 50 infectious units (i.e., 2000 particles) per cell (0), or mockinfected (A), was sedimented in parallel neutral sucrose gradients at 28,000rpm and 20” for 16hr. (B) 3Hlabeled Hirt supernatant DNA from cells infected with Ad2 (m.o.i. = 5.0) plus AAV(1.35) at 2000 narticles aer cell (0) was sedimented in neutral uradients as for panel A. 32P-labeledlinear duplex AAV DNA (Oj was added as a marker.
with AAV(1.35) or AAV(1.32) even using culture was analyzed in neutral sucrose up to 300,000 particles per cell in the gradients. Figure 4A shows the control presence or absence of helper adenovirus. experiment which demonstrates the autoIn contrast, AAV progeny strand DNA interference of AAV(1.41) particles at the level of AAV DNA replication. As the synthesis was detectable by sedimentation assay of Hirt supernatant DNA from cells multiplicity of AAV(1.41) was increased infected with adenovirus together with there was a decrease in 14.5 S DNA AAV(1.41) at a multiplicity of 0.02 (data synthesis and an increase in 10 S DNA not shown). This is equivalent to about synthesis. Over the multiplicity range em1 AAV(1.41) particle per cell. In the absence ployed, the total amount of AAV DNA of helper adenovirus there was no detectable synthesis (i.e., sum of 14.5 S + 10 S DNA) replication of any density class of AAV did not vary greatly. particles including AAV(1.41). Also, AAVThe viral DNA isolated from cells that (1.35) or AAV(1.32) DNA could not be were infected with AAV(1.41) particles at replicated when cells were coinfected with a multiplicity of 1.0 together with increasing either of these species together with helper amounts of AAV(1.35) particles is shown adenovirus and uv-inactivated AAV(1.41) in Fig. 4B. At relatively low levels of particles (see below). AAV(1.35) particles (350 to 3500 particles per cell or a ratio of 1.35:1.41 particles of about 9:l to 9O:l) there was progressive Interference with Replication of Standard inhibition of standard, 14.5 S DNA and a AAV(1.41) by AAV(1.35) or AAV(I 32) proportional increase in 10 S DNA synthesis. In the experiment shown in Figs. 4B At higher levels of AAV(1.35) particles and C, cells were coinfected with Ad2 at a (35,000 per cell or a ratio of 1.35:1.41 of m.o.i. of 5 PFUkell, AAV(1.41) particles 9OO:l) there was a large overall inhibition at a multiplicity of 1.0 (equivalent to about of AAV DNA synthesis including decreased 40 particles per cell), and varying amounts amounts of both 14.5 S and 10 S DNA. of AAV(1.35) or AAV(1.32) particles. In the Importantly, even at this level, the incontrol culture (Fig. 4A), cells were infected hibition of 14.5 S DNA synthesis was with Ad2 and varying amounts of AAV(1.41) proportionally greater than that of 10 S particles. 3H-Labeled, intracellular viral DNA when compared to the control with no DNA from the Hirt supernatant of each added AAV(1.35) particles. Thus, at least
170
ET AL.
LAUGHLIN
at lower levels of added AAV(1.35) particles, the inhibition resembled that seen in the autointerference experiment (compare Figs. 4A and B). These results suggest that 10 S DNA can be made in response to added AAV(1.35) particles but only in the presence of both adenovirus and AAV(1.41) virus as helpers. We conclude from these results that a helper function provided by AAV(1.41) is required for 10 S DNA production. The great inhibition of total AAV DNA synthesis seen when higher amounts of AAV(1.35) particles were added may be interpreted as inhibition of AAV(1.41) to the level at which it could supply insuf6cient helper function for 10 S DNA synthesis (see Discussion). In Fig. 4C are shown similar experiments using added AAV(1.32) particles. As seen in Fig. 4C, there was progressive inhibition of the standard 1.41 virus replication with
increasing amounts of added 1.32 particles through less than that seen with similar numbers of AAV(1.35) particles. Also, there was inhibition rather than stimulation (as in Fig. 4B) of 10 S DNA replication and no detectable stimulation of synthesis of any DNA of the size expected to be in 1.32 particles (about 3-6 S). In this respect the inhibition mediated by AAV( 1.32) particles did not appear to reflect directly that seen in autointerference experiments (Fig. 4A). These experiments suggest that the 1.35 and perhaps the 1.32 particles may be DI particles. However, the experiments did not reliably determine whether any function of the infecting 1.35 or 1.32 genome was necessary for interference or if interference was merely the result of blocking adsorption to the cells of AAV(1.41) particles. These questions were addressed in the experiments described in the following sections.
C
10
33
50
IO
30
50
10
30
50
FRACTION NUMBER
FIG. 4. Analysis of autointerference exhibited by AAV(1.41) particles and interference exhibited by AAV(1.35) and AAV(1.32) particles. Monolayers (75 cm2)of KB cells were infected with adenovirus 2 (3 PFUkell) and various multiplicities of AAV(1.41) and AAV(1.35) or AAV(1.32) particles. The Hirt supernatant DNA (labeled with [3H]thymidine from X-20 hr postinfection) was sedimented in neutral sucrosegradients. (A) Cells wereinfectedwithAAV(l.41) at multiplicities of0.2(A), 1.0 (O), or 10.0(O) infectious units/cell. (B) Cells were infected with AAV(1.41) at an m.o.i. of 1.0 infectious unit (40 particles)/cell, (0) or together with AAV(1.35) at 350 (O), 3500 (A), or 35,000 (A.) particles per cell. (C) Cells were infected with AAV(1.41) at an m.o.i. of 1.0 infectious unit (40 particles)/cell (0) or together with AAV(1.32) at 1000(O), 10,000(A), or 30,000(A) particles per cell. Note: The same gradient profile of DNA from cells infected with AAV(1.41) at an m.o.i. of 1.0 (0) is plotted in all three panels for comparison. This multiplicity represents approximately 40 AAV(1.41) particles per cell.
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
PARTICLES
171
DI-Mediated Inhibition of AAV is not Due to Inhibition of Cell&w Adsorption
a3 2.8 9.2 280 Li 16 53 160 + AAW 351 P.ruc,eE Added Pet Cell ,x,W) AAw1.32, Pamdes mded Per cdl h1w FIG. 5. Effect of AAV DI particles on cellular adsorption of AAV(1.41) and effect of uv irradiation on AAV DI inhibition. KB cells in monolayers were infected with Ad2 at a multiplicity of 5 together with AAV(1.41) and varying amounts of AAV(1.35) or (1.32) particles. (A) Cells were infected with 32P-labeled AAV(1.41) particles at a multiplicity of 200 infectious units (i.e., 8000 particles) per cell together with the indicated amounts of AAV(1.35) particles. The amount of 32P-labeled AAV particles adsorbed was measured as described under Materials and Methods and is expressed as a percentage of the amount bound in the absence of added AAV(1.35) particles [35% of total added 32P-labeled AAV(1.41) particles]. (B) An analogous experiment to that in A except that AAV(1.32) particles were used instead of (1.35) particles. (C) Cells were infected with Ad2 (5 PFlJl cell) and AAV(1.41) (3 infectious units/cell) together with the indicated amounts of AAV(1.35) particles. DNA was labeled at 16 to 20 hr postinfection with [3H]thymidine and extracted and analyzed on neutral sucrose gradients as described under Materials and Methods. The total amount of 14.5 S AAV DNA in gradients was calculated and plotted as shown. The data was corrected for background radioactivity in the analogous regions of a sucrose gradient of DNA from cells infected with Ad2 alone. The amount of 3H-labeled 14.5 S AAV DNA sythesized in the control in the absence of any added AAV(I.35) particles was 2.9 x lo4 countslmin. (@) AAV(1.35) particles not irradiated; (0) AAV(1.35) particles uv irradiated for 20 min. (0) Cells infected with Ad2 (5 PFU/cell), AAV(1.35) particles, and uv-irradiated AAV(1.41) particles (equivalent of 3 infections units/cell). (D) An analogous experiment to (C) except that AAV(1.32) particles were used in place of AAV(1.35) particles. (A) AAV(1.32) particles not irradiated; (V) AAV(1.32) irradiated for 20 min; (A) AAV(1.32) irradiated for 360 min. (C!) Cells infected with Ad2 (5 PFU/cell), AAV(1.32) particles, and uv-irradiated AAV(1.41) particles (equivalent of 3 infections units/cell).
32P-Labeled AAV(1.41) particles were adsorbed to KB monolayer cells at multiplicities of 200 infectious units per cell in the presence of varying amounts of AAV(1.35) or AAV(1.32) particles. It is difficult to perform this experiment with lower multiplicities of 32P-labeled AAV( 1.41) because of limitations on the specific activity of AAV DNA which can be obtained. The cell layer was then washed extensively and the proportion of cell bound 32P-labeled AAV(1.41) was determined (Figs. 5A and B). At the range of concentrations used, the 1.35 and 1.32 particles did not interfere with the proportion of AAV(1.41) particles bound [about 30 to 35% of the total AAV(1.41) particles]. However, as shown in Figs. 5C and D, similar amounts of AAV(1.35) and (1.32) particles showed inhibition of AAV 14.5 S DNA replication. These results suggest that inhibition by AAV DI particles is not mediated at the 1eveI of adsorption to the host cell. Effect of uv Irradiation
on AAV Particles
The effect of uv irradiation on the biological properties of the AAV particles was investigated and the results are summarized in the experiment shown in Figs. 5C and D. In this experiment, AAV particles were uv irradiated for 20 or 360 min. A ZO-min irradiation was sufficient to decrease the infectivity of AAV(I.41) by 105-fold. As shown in Figs. 5C and D, the inhibition of 14.5 S DNA synthesis by the 1.35 or 1.32 particles could be abolished by uv irradiation. This implies; (a) that some function, presumably replication or perhaps transcription, of AAV(1.35) or AAV(1.32) genomes was required in order to obtain interference and (b) that the inhibition was not mediated at the level of adsorption. A ZO-minute exposure to uv light completely destroyed all the DI activity of the 1.35 particles (Fig. 5C) whereas a 360-min exposure was required to inactivate most of the 1.32 particles (Fig. 5D). These observa-
172
LAUGHLIN
tions are qualitatively consistent with the relative target size for the DI activity of the 1.35 and 1.32 genomes expected on the basis of their molecular weights (de la Maza and Carter, 1978). We have not extended these data to provide a quantitative estimate of the target sizes. When KB cells were infected with adenovirus together with the uv-irradiated AAV(1.41) particles, there was no detectable peak of AAV DNA in the Hirt supernatant and the sedimentation profile was indistinguishable from that of an adenovirus-infected control culture (data not shown). Similar results were obtained when the cells were coinfected with adenovirus, uv-irradiated AAV(1.41) and either AAV(1.35) or AAV(1.32) particles (Figs. 5C and D). Again there was no detectable synthesis of AAV progeny strands of either standard or DI virus. This is consistent with the hypothesis suggested above, that replication of AAV DI particles requires a helper function provided by AAV(1.41) particles.
ET AL.
These experiments show that AAV(1.35) and (1.32) particles do not directly inhibit adenovirus in the absense of 1.41 particles and may not do so even in the presence of 1.41 particles. This latter conclusion is more difficult to be certain about because we cannot readily resolve two effects: (a) a direct effect of adenovirus inhibition mediated by replicating AAV(1.35) or (1.32) and (b) an indirect effect of inhibition of AAV(1.41) by AAV(1.35) or (1.32) which may lead in turn to a release of adenovirus inhibition mediated by AAV(1.41). DISCUSSION
The experiments reported here clearly establish the presence of defective-interfering (DI) particles of adeno-associated virus. We have shown that AAV(1.35) and perhaps AAV(1.32) particles, when added to KB cells together with both AAV(1.41) and Ad2 particles, inhibit production of both standard (infectious) AAV progeny and of standard 14.5 S AAV DNA molecules. Several observations indicate that this Effect of AAV Particles on Adenovirus inhibition is mediated by an intracellular Multiplication function of the AAV(1.35) or (1.32) DNA As shown in Table 3, neither AAV(1.35) genome rather than by the viral protein or nor AAV(1.32) particles showed any inhibi- at the level of adsorption of virus. The tion of the yield of infectious adenovirus at inhibitory activity was inactivated by uv an adenovirus multiplicity of either 10 or 1. irradiation. There was a qualitative corThe control experiment (Table 3) showed that relation between the uv dose required for the inhibition of adenovirus by AAV(1.41) inactivation of the inhibition and the size did occur as reported previously (Carter of the inhibitory genome. Inhibition required et al., 1979). a larger number of AAV(1.32) particles than The failure of 1.35 or 1.32 particles to AAV(1.35) particles which may reflect an block production of infectious adenovirus excess of truly empty AAV particles in the particles may be accounted for by the 1.32 population or the small size of the failure to block adenovirus DNA replication AAV(1.32) DNA genomes. The AAV parti(Table 4). In the control experiments, cles containing the largest aberrant genomes AAV(1.41) particles inhibited incorporation cannot be separated readily from AAV(1.41) of isotopic label into adenovirus DNA particles (de la Maza and Carter, 19’78)but (Table 4) and the AAV(1.35) and AAV(1.32) we conclude by analogy that these aberrant preparations inhibited labeling of AAV- virions may also be DI particles. Thus, the (1.41) 14.5 S DNA (not shown). When autointerference exhibited by AAV(1.41) AAV(1.35) or (1.32) particles were added particle preparations (Carter et al., 1979; to cells, together with adenovirus and see also Fig. 5A) is postulated to be AAV(1.41) particles, there was no addi- mediated by DI particles. The AAV DI particles appear to satisfy tional inhibition, or in some cases even less inhibition, of adenovirus multiplica- all the criteria of DI particles listed by tion over that achieved by the AAV(1.41) Huang and Baltimore (1970, 1977). AAV particles alone (Tables 3 and 4). DI particles have predominantly lighter
ADENO-ASSOCIATED
VIRUS DEFECTIVE-INTERFERING
PARTICLES
173
densities in CsCl than standard AAV, for progeny strand DNA production of contain aberrant, deleted genomes (de la standard AAV. Maza and Carter 1978), inhibit replication The above discussion assumes that the of standard genomes intracellularly, and infecting AAV DI genome is in fact repliappear to require a helper function supplied cated. The uv inactivation experiments and by standard AAV (see below). The DI enhancement of DI DNA production by genomes accumulate preferentially at high added DI particles support this assumption multiplicity of infection (Fig. 4A), and DI but do not constitute rigorous proof. An particles accumulate rapidly during high alternative explanation of the above data is multiplicity undiluted passage (M. Myers, that DI genomes might be produced directly, C. A. Laughlin, D. L. Risin, unpublished and only, from standard genome templates experiments). by an aberrant replication. Added exogenous The mechanism of inhibition by AAV DI DI genomes might somehow enhance this particles is not clear. However, the present process by a mechanism which does not results argue that replication of the AAV DI involve use of the added DI genome as a genome is required. The AAV DI genomes replication template. Experiments designed like other viral DI genomes (Huang and to obtain direct proof of replication of added Baltimore, 1977) are enriched for the region DI genomes are currently in progress. We do not know if all or only some of containing the replication origin (Hauswirth and Berns, 1978; de la Maza and Carter, the aberrant genomes in light-density AAV 1978) and thus should be able to replicate. particles are DI molecules. For instance, The results presented here suggest that the genomes in 1.35 g/cm3 particles appear production of AAV DI genomes requires to be topologically heterogeneous and only some are snap-back molecules, whereas a helper factor supplied by standard AAV. Thus, AAV DI genome production is those from the 1.41 or 1.32 particles appear detected only in cells jointly infected with topologically more homogeneous and are DI genomes [or a high multiplicity of AAV- nearly all snap-back molecules (de la Maza (1.41) particles], AAV(1.41>, and Ad particles. and Carter, 1978). More interestingly, some DI genome replication was not detected in aberrant genomes contain either left- or cells infected only by DI particles or in the right-hand terminal regions of the AAV presence of Ad2, or Ad2 together with genome (de la Maza and Carter, 1978) and uv-inactivated AAV(1.41). Also, AAV DI we do not know whether both classes are genomes inhibit AAV replication but do not DI particles. For any DI inhibition which appear to inhibit adenovirus replication. occurred at the level of transcription it is The simplest interpretation of these data possible that only the left-hand end molecules is that in addition to the adenovirus would be inhibitory since this is the probable helper factors for AAV, the production or location of the AAV promoter (Carter replication of progeny AAV DI particles et al., 1976; Jay et al., 1978). requires an AAV-specified helper function. This function must be either one or more of ACKNOWLEDGMENTS the AAV capsid polypeptides or the two We thank R. Friedman for critical review and Billie additional polypeptides generated during posttranslational cleavage of these proteins Healy for expert preparation of the manuscript. since these are the only known products of translation of AAV mRNA (Buller and REFERENCES Rose, 1978). The AAV DI genomes should not produce these proteins because they BULLER, R. M. L., and ROSE,J. A. (1978). Characterization of adeno-associated virus polypeptides syncontain deletions in the codigenic regions thesized in viva and in vitro. In “Replication of of the DNA (Carter et aZ., 1976; de la Maza Mammalian Parvoviruses” (D. C. Ward and P. and Carter, 1978). On the basis of these Tattersall, eds.) pp. 399-410. Cold Spring Harbor. results, we also argue that it is likely Laboratory, Cold Spring Harbor, N. Y. that an AAV capsid polypeptide is required CARTER, B. J., FIFE, K. H., DE LA MAZA, L. M.,
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and BERNS, K. I. (19’76). Genome localization of adeno-associatedvirus RNA. J. Viral. 19,1044- 1053. CARTER, B. J., KOCZOT,F. J., GARRISON,J., ROSE, J. A., and DOLIN, R. (1973). Separate helper functions provided by adenovirus for adenovirusassociated virus multiplication. Nature New Biol. 244, 71-73. CARTER, B. J., LAUGHLIN, C. A., DE LA MAZA, L. M., and MYERS, M. (1979). Adeno-associated virus auto-interference. Virology 92, 449-462. DE LA MAZA,.L. M., and CARTER, B. J. (1978):DNA structure of incomplete adeno-associated virus particles. In “Replication of Mammalian Parvoviruses” (D. C. Ward and P. Tattersall, eds.), pp. 193-204. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. GERRY, H. W., KELLY, T. J., JR., and BERNS, K. I. (1973). Arrangement of nucleotide sequences in adeno-associated virus DNA J. Mol. Biol. 79, 207-225. HAUSWIRTH, W. W., and BERNS, K. I. (1978). Initiation and termination of adeno-associated virus DNA replication. In “Replication of Mammalian Parvoviruses” (D. C. Ward and P. Tattersall, eds.), pp. 257-267. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. HUANG, A. S., and BALTIMORE, D. (1970). Defective viral particles and viral disease processes. Nature (London)
226, 325-327.
HUANG, A. S., and BALTIMORE, D. (1977). Defective interfering animal viruses. In “Comprehensive
ET AL.
Virology” (H. Frankel-Conrat and R. R. Wagner, eds.), Vol. 10, pp 73-116. Plenum, New York. JAY, F. T., LAUGHLIN, C. A., DE LA MAZA, L. M., CARTER,B. J., and COOK, W. (1978). Adenoassociated virus RNA synthesis in wivo and in vitro. In “Replication of Mammalian Parvoviruses” (D. Ward and P. Tattersall, eds.) pp. 385-379. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. KOCZOT,F. J., CARTER, B. J., GARON, C. F., and ROSE,J. A. (1973). Self-complementarity of terminal sequences within plus or minus strands of adenovirus-associated virus DNA. Proc. Nat. Acad. Sci. USA 70, 215-219. LOWRY, 0. H., ROSEBOROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. ROSE,J. A. (1974). Parvovirus reproduction. In “Comprehensive Virology” (H. Frankel-Conrat and R. R. Wagner, eds.), Vol. 3, pp. 1-61. Plenum, New York. ROSE, J. A., MAIZEL, J. V. JR., INMAN, J. K., and SHATKIN, A. J. (1971). Structural proteins of adenovirus-associated virus. J. Viral. 8, 766-770. SIEGL, G. (1976). “The Parvoviruses.” SpringerVerlag, Wien/New York. STRAUSS, S. W., SEBRING, E. D., and ROSE, J. A. (1976). Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc. Nat. Aead. Sci. USA 73, 742-746. TATTERSALL, P., CAWTE, P. J., SHATKIN,A. J., and WARD, D. C. (1976). Three structural polypeptides coded for by minute virus of mice, a parvovirus. J. Viral.
20, 273-289.