Isolation of intracellular replicative forms and progeny single strands of DNA from parvovirus KRV in sucrose gradients containing guanidine hydrochloride

VIROLOGY

76,

464-467

(1977)

isolation of Intracellular Replicative Forms and Progeny Single Strands of DNA from Parvovirus KRV in Sucrose Gradients Containing Guanidine Hydrochloride’, 2 GEORGE Biology

Division,

Oak

Ridge

LAVELLE

National Laboratory of Biomedical Sciences, Accepted

AND

ANNA

and University Oak Ridge, September

TAI LI

of Tennesseeauk Tennessee 37830

Ridge

Graduate

School

28,1976

When cells infected with Kilham rat virus (KRV) were incubated in 4 M guanidineHCl, the single-stranded DNA was completely released from virions of KRV, but doublestranded DNA was not denatured. The three forms of viral DNA released had properties, respectively, of double-stranded replicative-form DNA, single-stranded progeny DNA, and an intermediate DNA species. Sucrose gradients containing 4 M guanidine-HCl were used to isolate these intracellular DNA forms with complete separation from high molecular weight cellular DNA. After a short pulse, radioactivity in low molecular weight DNA was found almost exclusively in molecules having sedimentation properties of viral replicative-form DNA. During chase incubation, this radioactivity progressively shifted to the position of single-stranded progeny DNA.

Evidence that synthesis of the singlestranded DNA of parvoviruses (molecular weight 1.5-1.7 x lo’? (1) is mediated by a double-stranded replicative form (RF) is supported by recent reports from several laboratories (2-6 1. Double-stranded, pulse-labeled molecules with properties of viral RF have been recovered from cells infected with the parvoviruses H-l (2) LuIII (3), minute virus of mice (MVM) (4, 5), and Kilham rat virus (KRV) (Li and Lavelle, unpublished observations) by the selective extraction method of Hirt (7). Observations on the synthesis of progeny single strands of DNA have been limited, hcwever, partly because these are not recovered from cells by the Hirt method, possibly because progeny DNA is encapsidated as quickly as it is made and is precipitated with the Hirt pellet. In support of

this theory, our experience has been that Hirt extraction does not release DNA from purified vii-ions. To facilitate studies of progeny single-strand synthesis, we use a method, described here, which permits isolation of intracellular RF and progeny DNA of KRV with excellent separation from high molecular weight cellular DNA. The method employs lysis of cells by guanidine-HCl and sedimentation of the lysate in sucrose gradients containing guanidine-HCl. The utility of the method is demonstrated by results which show production of single-stranded progeny DNA from replicating intermediates in pulsechase experiments. As shown in Fig. lA, when DNA was extracted from purified KRV by incubation in 4 M guanidine-HCl, it cosedimented with marker KRV DNA in sucrose-guanidine-HCl gradients. The marker DNA was extracted from purified virus using protease-detergenkphenol methods (8, 9). Thus, DNA was completely released by this extraction method. Our preliminary studies indicated that KRV DNA, which is single-stranded (10,

1 By acceptance of this article, the publisher acknowledges the right of the U.S. Government to retain a nonexclusive royalty-free license in and to any copyright covering the article. 2 This work was sponsored by the U.S. Energy Research and Development Administration under contract with Union Carbide Corporation. 464 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

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rate of 25 S, relative to the double-stranded form of adenovirus-associated virus (AAV) DNA (8), which has a rate of 14.5 S in sucrose gradients containing 1 M NaCl (J. A. Rose, personal communication). Figure 1B shows that double-stranded AAV DNA and KRV DNA maintained these relative sedimentation rates in gradients which contain 4 M guanidine-HCl. These results suggested that doublestranded RF and single-stranded progeny DNA of KRV could be isolated simultaneously from infected cells, provided they were released from nuclei without shearing cellular DNA. The problem of shear is especially important, because RF is replicated only in cells which are synthesizing cellular DNA (12). Thus, the proportion of radioactivity which is found in viral-specific DNA after a pulse with radioactive thymidine (dThd) is small, and fragmented cellular DNA readily masks the species of DNA under study. Treatment of cells with guanidine-HCl released low molecular weight viral DNA without shear, as shown by the results in Fig. 2. Monolayers of normal rat kidney cells (13) were infected with KRV (14) for 23 hr. Infected and control cultures were pulsed with radioactive dThd for 10 min and chased for 1 or 8 hr in medium containing excess unlabeled dThd. Cells were removed from culture dishes and layered onto gradients of 5-20% sucrose containing 4 M guanidineHCl. The loaded gradients were incubated overnight at room temperature to allow lysis of cells and encapsidated virus. Figure 2A and B shows sedimentation profiles obtained after a lo-min pulse with [“HlThd. The internal marker is “2P-labeled KRV DNA from purified virus. In Fig. 2A, 80% of the total acid-precipitable counts per minute were found in high molecular weight DNA in the bottom two fractions of the gradient. A small peak of radioactivity appeared at the 16 S region (arrow). When only low molecular weight DNA was considered, by omitting the two bottom fractions, the profile shown in Fig. 2B was obtained. The peak sedimentation for low molecular weight 3H-labeled DNA was at approximately 16 S, while that for

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FIG. 1. Sedimentation of KRV DNA in sucrose gradients containing 4 M guanidine-HCl. (A) Eightmolar guanidine-HCl was added to an equal volume of [3H1dThd-labeled, CsCl-purified KRV (5.3 x lo4 cpm), and the mixture was incubated at room temperature for 20 min and layered on a 520% sucrose gradient containing 4 M guanidine-HCl, 10 mM Tris-HCl (pH 8.01, 1 m&f EDTA. :‘2P-Labeled KRV DNA (5 x lo* cpm) extracted from purified virus by protease-detergent-phenol (8, 9) was added as marker. Sedimentation was at 42,000 rpm for 3 hr at 20” in the SW 50.1 rotor of a Beckman ultracentrifuge. (B) 3H-Labeled KRV DNA (1.4 x 10” cpm) was sedimented with double-stranded 14C-labeled DNA of AAV (8.5 x lo2 cpml. AAV type 2 (with adenovirus type 2) was obtained from Dr. James Rose, NIH. Production and purification of virus, and extraction and purification of viral DNA were carried out exactly as described (8, 9). Composition of the gradient was the same as in (A). Sedimentation was at 39,000 rpm for 6 hr at 20” in the Beckman SW 41 rotor. Sedimentation is from right to left in both A and B.

marker KRV DNA was at approximately 25 S. The 16 S DNA has several properties of viral RF: by electron microscopy it was largely double-stranded, was of viral unit length, and contained DNA complementary to virion DNA by molecular hybridization (Li, Lavelle, and Tennant, manuscript in preparation). The observed sedi-

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FIG. 2. Isolation of intracellular forms of KRV DNA in sucrose gradients containing 4 M guanidine-HCl. Subconfluent monolayers of normal rat kidney cells (l-l.5 x lo6 cells/lOO-mm plastic culture dish) (13), propagated in McCoy medium supplemented with 10% fetal calf serum and 2 m&f glutamine, were infected with l-10 PFU/cell of KRV (14). After 23 hr of infection, cultures were pulselabeled for 10 min with 100 &i/ml of [3H1dThd (54 Ci/mmol) in 5 ml of medium, washed, and incubated in medium containing 1 x 10m4 M unlabeled dThd. After 10 min of pulse and 1 and 8 hr of chase incubation, cells were removed from culture dishes by 2- to 3-min treatment with 0.25% trypsin containing 1 m&f EDTA followed by gentle scraping and pipetting. Five x IO4 to 1 x lo5 cells in 0.3 ml were layered onto 11.5-ml sucrose gradients containing 4 M guanidine-HCl, as in Fig. 1. Two-hundred micrograms of proteinase K (EM Laboratories, Inc., Elmsford, N.Y.) were added, and the loaded gradients were incubated at 20” overnight (lo-18 hr) prior to sedimentation. Centrifugation was at 39,000 rpm for 7 hr at 20” in the Beckman SW 41 rotor. Fractions of 0.25 ml were collected, and the acidprecipitable counts per minute were determined. Sedimentation is from right to left in all cases. (A, B) A lo-min pulse. In (A), percentage counts per minute per fraction was determined for the entire gradient; in (B), the bottom two fractions were omit-

mentation rate was similar to that reported for RF DNA of H-l (2) and LuIII (3) parvoviruses . Figure 2C and D shows that after 1 and 8 hr of chase incubation, respectively, in the presence of excess unlabeled dThd, increasing proportions of low molecular weight radioactivity cosedimented with marker virion DNA (25 S), while the percentage of counts per minute in 16 S DNA decreased. RF DNA of parvoviruses, like that of bacteriophages having singlestranded genomes, is a duplex of viral and complementary strands (2,3,15). Because complementary DNA is not encapsidated into KRV vu-ions (16, 171, only the viral strand of the labeled duplex would be expected to appear in single-stranded progeny during a chase incubation. In agreement with this pattern of progeny synthesis, Fig. 2 shows that only part of 16 S DNA was found to chase into the position of progeny DNA (25 S). Thus, the results of the above experiment strongly support the conclusion that 16 S DNA includes intermediates in the synthesis of progenystrand DNA. A third species of DNA was always found at a position intermediate between 16 and 25 S as a leading shoulder on the 16 S peak (Fig. 2B-D). Further analysis of this DNA by Sl-endonuclease digestion indicated that it was partly single-stranded (Li, Lavelle, and Tennant, manuscript in preparation). The proportion of DNA in this species also decreased with time of chase. Hence, this DNA had some properties of replicative intermediates (RI). The three species of DNA described here were never found in uninfected control cells. More complete analyses of their properties are in progress and will be reported subsequently. The use of guanidine-HCl extraction should permit quantited from calculations of percentage counts per minute. (Cl One hour and (D) 8 hr of chase incubation. Calculations of percentage counts per minute are as in (B). Gradients contained 3-5 x lo4 3H cpm. Internal marker is 32P-labeled KRV DNA from purified virus (1.4 x lo3 cpm/gradient) which was added immediately prior to sedimentation. The arrow in (A) indicates the approximate 16 S position. Closed circles, 3H; open circles, 32P.

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tative kinetic studies of RF replication, progeny-strand synthesis, and structural studies of RF and RI. The method has the additional advantages of being single-step and providing excellent separation from high molecular weight cellular DNA. It is also noteworthy that broken or degraded DNA of very low molecular weight was never found in the upper fractions of gradients under these extraction conditions. The method has the following limitations: (i) The number of cells which can be extracted without overloading gradients with high molecular weight cellular DNA is limited, which limits the amount of each species which can be recovered; the numbers of cells used in Fig. 2 were found to be suitable for 11.5ml gradients. (ii) The distribution of radioactivity in favor of high molecular weight cellular DNA is so great that relating counts per minute per gradient to counts per minute in viral DNA is inexact. (iii) Because infected cells are growing cells, their numbers change with time of chase, as do also the counts per minute of viral DNA per cell. For these reasons we compare, as in Fig. 2, the relative proportions of radioactivity in low molecular weight DNA species. DNA extracted from purified adenoviruses by guanidine-HC1 appeared to consist of DNA complexed with protein which contributed to formation of circular molecules (18). The complexed DNA had a sedimentation rate different from that of protease-detergent-extracted viral DNA. In our studies of KRV DNA isolated by guanidine-HCI, we have not found evidence of a bound protein. The sedimentation rate of viral DNA extracted by guanidine-HCl was identical to marker DNA under the conditions shown in Fig. 1, where neither proteases nor detergents were added. With respect to intracellular DNA isolated un-

der the conditions of Fig. 2, proteins labeled with a radioactive amino acid did not cosediment with viral DNA. Nevertheless, we routinely extract viral DNA species with detergent and phenol (19) prior to biochemical and biophysical analyses. REFERENCES

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