Archives of Virology

Archives of Virology 59, 331--343 (1979)

© by Spmnger-Vertag 1979

Macromoleeular Structure of Nuclear Polyhedrosis Virus Genome By L. I. STROKOVSKAYA,I. N. SKURATOVSKAYA,A. P. GUDZ-GORBAN, E. N. Z~ER~BTSOVA, V. I. I)~I~A, and I. P. KoK institute of Molecular Biology and Genetics of the Ukrainia:n S.S.R., Kiev, U.S.S.R. With 8 Figures Accepted September 27, 1978

Summary DNA preparations from nuclear polyhedrosis virus (NPV) of Galleria mellonella L. (GmL) were fractionated in high ionic strength neutral sucrose gradient. This procedure allowed a separation of snpercoiled infectious DNA molecules with contour length of 48--52 bL from infectious open ring DNA molecule, and noninfectious linear DNA molecules of the same size. In addition a heterogeneity of supercoiled DNA molecules was detected. Covalently closed DNA molecules did not contain protein or ribonucleotide ligands which could be digested by pronase or pancreatic I{Nase treatment. I t is concluded from data on the infectivity of different molecular forms of DNA and reassociation kinetics studies, that the genome of G m L NPV is a unique ring nueleotide sequence with a molecular weight of about 90--100 × l06.

Introduction Nuclear polyhedrosis viruses (NPVs) belong to the Baeuloviridae family. Studies on insect viruses, especially NPVs have become increasingly important, The potential use of NPVs as natural pest control agents is currently tmder investigation. Studies of the structural and biological characteristics of viral genomes are also of great interest as they provide data which m a y lead to an understanding of the molecular basis of virus reproduction. The DNA of two NPVs have been shown to be heterogenous in their molecular forms and sizes. Infectious DNA preparations from Bombyx mori L. NPV contained ring molecules with a molecular weight of about 100 × 10 s (8, 9). infectious DNA from Galleria mellonella L. (GmL) NPV has also been shown to be heterogenous and to contain similar molecules (12).

0304-8608/79/0059/0331/$ 02.60

332

L . I . STI~OKOVSKAY£et al. :

I n f e c t i v i t y of large supercoiled N P V D N A molecules w i t h a molecular weight of a b o u t 100 × 10¢ has been d e m o n s t r a t e d (17). H o w e v e r , this finding does n o t resolve t h e p r o b l e m a b o u t t h e size a n d s t r u c t u r a l characteristics of t h e N P V genome, since infective D N A p r e p a r a t i o n s containing b o t h open circular molecules (also w i t h a molecular weight of a b o u t 100 × 106) a n d linear moleeules of different l e n g t h h a v e been found. T h e a i m of this s t u d y was to i d e n t i f y t h e s t r u c t u r a l characteristics of the G m L N P V genome. Centrifugation in high ionic s t r e n g t h n e u t r a l sucrose d e n s i t y g r a d i e n t s e p a r a t e d snpercoiled D N A molecules f r o m o t h e r circular molecules of t h e same size a n d f u r t h e r m o r e d e m o n s t r a t e d struetm'al h e t e r o g e n e i t y in t h e supercoiled molecules. I n f e c t i v i t y tests a n d D N A reassociation kinetics d a t a were used to d e t e r m i n e t h e g e n o m e size of G m L N P V .

Materials and Methods Virus and Insects The M-I strain of GmL N P V was used (17). The insect larvae used and their method of infection with N P V have been described previously (17).

Isolation and Puri]ication o/~H-Thymidine Labelled GmL N P V 3H-thymidine was injected into larvae (4.0 ~Ci/larva) 24, 48, or 72 hours after N P V infection (insects were dead by 100 hours after virus inoculation). Non-occluded virus particles were purified from dead larvae containing polyhedra as described previously (t7, 19). An additional purification in CsC1 gradients was done for virus preparations, in which the D N A was to be used in reassociation kinetic experiments. These virus preparations were eentrifugated in preformed CsC1 density gradients (1.2---1.5 g/em 3) at 25,000 rpm for 18 hours at 5 ° C in a SW 27, Spinco L 2. The gradients were fraetionated, and the E~60, radioactivity, and infectivity of each fraction were determined. Aliquots of each fraction were also taken for electron microscopic investigation. Freshly purified virus, lyophilized virus (from water suspensions), and virus stored at -- 20 ° C in 40 per cent sucrose solution, were used to determine the influence of virus storage on the presence of supereoiled D N A molecules. aH-DNA Puri/ication From GmL N P V D N A was extracted with phenol-detergent or detergent-salt (6) as previously described (17). Crystalline CsC1 (final concentration 1.t g/ml) was added to supernatants for protein flotation when D N A was extracted with detergent-salt. D N A preparations were eentrifugated for 30 minutes at 14,000 rpm in a K-24 centrifuge (Janetzki) at 5 ° C and then dialysed against 1 × SSC containing 10-a ~ EDTA. I n some preparations phenol t r e a t m e n t was accompanied by rigorous shaking at. room

temperature. Enzyme Digestion o / D N A For pronase digestion, D N A (50 ~xg/ml) was incubated with pronase (100 ag/m]) for 1 hour at 37 ° C. RNAse ("Sigma") for digestion experiments was heated a~ 100 ° C for i0 minutes, D N A (50 ~g/m]) was then incubated with RNAse (10 ~g/m]) for 30 minutes at 37 ° C. Centri]ugation o] D N A in Sucrose Density Gradients D N A solutions (10--15 ~g DNA) were layered onto 5---20 per cent sucrose gradient. Sucrose solutions were made in 1 M NaC1, 10-3 ~ E D T A , 10 2 ~ Tris-ttC1 p i t 7.5. Centrifugation was for 3 hours at 30,000 rpm at 18 ° C in a Spinco L2, SW40. After cen?Grifugation the tubes were pierced and 0.35 ml fractions were collected. Aliquots

Macromolecular Structure of Nuclear Polyhedrosis Virus Genome

333

of each fraction were taken for the determination of radioactivity, infectivity, and sucrose density. Peak fractions were dialysed against 1 × SSC containing 10-a M E D T A and used for electron micrscopic studies. Radioactivity and infectivity determinations, thermal denaturation and electron microscopic procedures have been described previously (17). :Phage X DNA was used as a marker for electron microscopy.

Centri/ugation o/D2~A in CsCl°EthBr Density Gradients Centrifugation of DNA in CsC1-EthBr density gradient was done as previously described (17). Fluorescent bands were collected with a syringe and dialysed overnight against 1 × SSC containing 10-~ M EDTA. These bands were then used in sucrose gradient centrifugation experiments.

Reassociation Kinetics o/GmL N P V D N A NPV and E. coli DNA preparations were dialyzed against 0.12 ~ Na-phosphate buffer solution, pH 6.8, concentrated to about 300 ~g/ml and then fragmented in a MSE sonic desintegrator. The average fragment size was shown by electron microscopy to be about 4.6 × 10'5. Thermal denaturation and reassociation of DNA molecules were done in 1 m m thermostated cuvettes of a "Unicam SP-8000" spectrophotometer. The temperature was measured using a copper-constatan thermocouple in the control cuvette. Temperature and opticM density data were registrated automatically. During denaturation the temperature was regularly increased in the euvette by 1° C per minute. When the optical density curves reached t/heir plateaux, the temperature in the cuvette was quickly lowered to 61.5 ° C. This temperature was 25 ° C lower than the Tra of GmL NPV DNA and was optimal for DNA reassoeiation.

Results Optimal Time o/"~H-Thymidine Incorporation Into Virus D N A 3 H - t h y m i d i n e was injected into the larval hemocoel different times after virus infection. The most highly labelled D N A was o b t a i n e d from larvae in which 3I-I. t h y m i d i n e had been injected 72 hours after infection. The specific a c t i v i t y of these D N A preparations was 1800--2000 cpm/~zg, which was 40-----50 times higher t h a n in preparations in which the 3I-I-thymidine had been injected 24 hours after infection. DlgA preparations isolated h'om insects injected with 3 H - t h y m i d i n e 72 hours after infection with N P V were used in the following experiments.

E//ect o/ Virus Storage on, D 5 \ 4 Structure D N A was extracted b y the H i r t m e t h o d from freshly purified virus, Iyophilized virus and virus stored in 40 per cent sucrose solution at - - 2 0 ° C, in order to determine the effect of virus storage on D N A structure. Supercoiled D N A molecules (17) were found in D N A preparations isolated from freshly purified a n d lyophilized virus. However, no supercoiled D N A molecules were observed in virus p r e p a r a t i o n s stored at - - 2 0 ° C.

D N A Fractionation D N A prepared from freshly purified virus isolated b y the H i r t or phenoldetergent m e t h o d w i t h o u t rigorous shaking, separated into four peaks in sucrose d e n s i t y g r a d i e n t (Figs. 1A, B). A major peak (III) appeared in the middle of the gradient, two m i n o r peaks (I a n d I I ) were observed near the b o t t o m of the gradients, a n d a fourth m i n o r peak (IV) was detected near the top of the gradients. 22 Arch.Virol. 59/4

334

L . I . STROKOVSKAYAet

al. :

The amount of radioactivity in these peaks varied according to the method of DNA isolation used. Peak I, I I and I I I were more highly labelled in DNA preparations isolated using the Hirt procedure than the phenol-detergent procedure. The bulk of radioactivity in DNA isolated using the phenol-detergent procedure appeared in the peak I I I (up to 60 per cent), while often no radioactivity appeared in peaks I and IV. Non-labelled UV-adsorbing material was detected at the top of the gradients.

! E

I

/I

qOO

L

300 700 200

,' /\

700 ,

l',l

,ffl / ',I

/

~.

I \ //^ \J : j I

5

/0

t~k

/3ZO gd f T,:JG,//Oi7 ,9Un76 CP

30

/ "~

. ..~-/~'x,

.5

70

/\ 50 ~

I X

7.5 20 ZS/cP3c/lon 17UiTIL~C P

30

Fig. 1. NPV DNA profiles in 5--20 per cent. neutral sucrose density gradients : A DNA extracted by the phenol-detergent method. B DNA extracted by the tIirt procedure I)NA infectivity (per cent of NP diseased insects). -- -- -- radioactivity (epm)

Infectivity tests demonstrated that for both DNA extraction procedures peaks I, II and III were ahvays infectious, and that peak IV was non-infectious (Figs. 1A, B).

Electron microscopic investigation revealed the presence of circular molecules with contour length of 48--52 V in peaks I and II. Peak llI contained also circular molecules (50--60 per cent) and linear molecules of the same contour lengths. In all these fractions short linear molecules (3--8 per cent) with contour length of i--i0 ix were seen. Circular molecules were observed in peaks I, II and III (Figs. 1 A, B). We have used three methods to explain such results : i. The two bands obtained from CsCl-EthBr gradient centrifugation were centrifuged separately on sucrose gradients (see above and 17). The lower band, containing mostly snpercoiled and open ring molecules produced peaks corresponding to peaks I, II and III (Fig. 2A); and the upper zone, consisting of open circular and linear molecules, produced peaks corresponding to peaks III and IV (Fig. 2B). W~e conclude that peaks I and I I contain supercoiled covalently closed DNA molecules, and that peak I I I consists of open circular nicked molecules. 2. The sucrose gradient peaks containing DNA molecules were studied by electron microscopy before and after Eth-Br treatment. Before dye treatment

/¢iaeromolecular Structure of Nuclear Polyhedrosis Virus Genome

335

p e a k I contained rigid circular molecules w i t h large loops (Fig. 3A), whilst in p e a k I I the molecules h a d m a n y crosses a n d small loops (Fig. 3B). The circular D N A molecules of p e a k I I I were t y p i c a l for open ring D N A structures (Fig. 3 C). On t r e a t m e n t w i t h E t h B r (100 ~g/ml), the circular molecules of p e a k s I a n d I I a p p e a r e d as t y p i c a l supereoiled molecules (Fig. 3D). H o w e v e r t h e open circular D N A molecules of p e a k I I I d i d n o t change their configuration on E t h B r treatment.

300 [

300

ZOO

700

I

I

7O

I

I

2O

~

I

I

3O

I

7O

F:3clio:7 RL/:be:

I

l

20

I

I

I

JO

/':~c/:'onRL:mbe:

Fig. 2. Profiles in 5--20 per cent neutral sucrose density gradients of N P V D N A bands from EthBr-CsC1 density gradients A lower band. B upper band

3. Only supercoiled infective D N A molecules of G m L N P V keep their i n f e c t i v i t y after t h e r m a l d e n a t u r a t i o n (17). The d a t a shown in Table 1 show t h a t the i n f e c t i v i t y in p e a k s I a n d I I was r e t a i n e d after t h e r m a l d e n a t u r a t i o n , b u t the i n f e c t i v i t y in p e a k I I I was lost. P e a k s I a n d I I were shown to contain e o v a l e n t l y closed molecules of t h e same size, b u t of different configuration. P e a k I I I consisted of open circular a n d linear molecules of t h e same length. R N A s e a n d p r o n a s e t r e a t m e n t of t h e D N A p r e p a r a t i o n s did n o t change their p r o p e r t i e s in sucrose gradients.

Table 1. I n / e e t i v i t y o/ D N A /rom neutral sucrose gradient density /raetions be]ore a n d a/ter thermal denaturation

Per cent of nuclear polyhedrosis after injection into larval hemocoel. D N A preparations were preheated before injection at Fraction No.

68 ° C (30 minutes) and cooled at room temperature

100 ° C (10 minutes) and quenched in ice

I II III IV

17 83 83 0

21 50 0 0

22*

336

L . I . STROKOVSKAYAe t

at. :

Fig. 3 A

Fig. 3 B Fig. 3. Electron microphotographs of N P V D N A from four peaks obtained after neutraI sucrose (5--20 per cent) density gradient eentrifugation: A D N A molecules of the first peak (Fig. 1B). B D N A molecules of the second peak (Fig. 1B). C circular D N A molecules from the third fraction (Fig. 1B). D D N A moleeutc from the second peak in the presence of excess E t h B r

Macromolecular Structure of Nuclear Polyhedrosis Virus Genome

Fig. 3 C

Fig. 3 D

337

338

L. I, STttOKOVSKAYA e t a l . : S t a b i l i t y o/ V i r a l D N A

We compared the sedimentation characteristics in sucrose gradients of DNA extracted with (Fig. 4) or without (Fig. 1) rigorous shaking. On rigorous shaking, radioactivity increased in the low density peak and decreased in the high density peaks. The radioactivity in peak I I I containing up to 80 per cent circular molecules, remained after DNA extraction with rigorous shaking. Figure 5 shows the distribution of the sizes of the DNA molecules from each peak of Figure 4. Peaks IVA, ]B, C contained linear molecules with contour length up t,o about 20--25 p.. DNA infectivity was demonstrated in peak I I I using i n vivo tests. The supercoiled

,AA/t ,,,i!vvi J~

10

75 20 2J i-/'aol/on nu/~e/"

dO

700

gd~

Fig. 4. NPV DNA profile in a 5----20 per cent sucrose gradient; DNA was obtained using the phenol-detergent method with rigorous shaking .... radioactivity (epm). infectivity (per cent of NP-diseased larvae)

10 152025

S 10 Z4

~'0~550

d/O 50 ZenGth f/z/z)

5 70 78 2025J0

40

b-70 20 30 Length (/z~)

Fig. 5. Size distribution of DNA molecules in peaks No. I I I , IVa, I V b and IVe from Fig. 4

Maeromoleeular S~ructure of Nuclear Polyhedrosis Virus Genome

339

molecules of peaks I and I I were also infectious although less material was found there. The linear molecules from psak IV, of contour length up to 20--25 ~, were non-infectious. These data confirm t h a t infectious circular molecules are found in D N A prepared using "rigorous" conditions. G m L N P V Genome Si~e

We have above mentioned t h a t non-labelled UV-adsorbing material is found a~ the ~op of neutral sucrose density gradient of purified N P V DNA. Virus preparations were purified in CsC1 density gradients in order to eliminate this material (see above). After centrifugation in CsC1 all the labelled material was pelleted and non-labelled material was observed in the gradients (Fig. 6). The pellet contained no infectious virus particles and consisted of nueleoprotein fibrils, and cireular and linear D N A molecules. This pellet was used for D N A isolation. CsCI-pt~rified D N A usually banded as a wide peak of radioactivity (Fig. 7) in

3.70 3

Z.~d

20

7.70d

zO

S iO iS fpeo//or F u m 6 s n

Fig. 6. CsCt density gra~dient eentrifugation of N P V infecting GmL. CsC1 = 1.2--1.5 optical density (E2a0). -- -- -- radioactivity (epm) -

-

6"00

17oo%

itJ

zlO0 i

)

I

200

~

~ i/

I0

£0 FneoiloR iTumbeP

d0

Fig. 7. DNA profiles in 5--20 per cent neutral sucrose density gradients. DIN-A was extracted from virus material purified on CsCl density gradient (Fig. 6) ............. radioactivity (epm). -- -- -- infectivity (per cent of NP-diseascd larvae)

340

L . I . SW~OKOVS~:AYAet a l . :

sucrose density gradients. No UV-adsorbing material was found on the top of gradient. Thermal denaturation of CsCl-purified N P V DNA was studied. The width of the melting interval (in 1 × SSC) was 12 ° C and the Tm was 86.5 ° C (corresponding to 40 per cent GC). The h)Terchromicity was about 30 per cent and no D N A denaturation occurred during sonication.

J

z

I

o

[

I

1

I

f

T

O,S

I

~

I

f

zo

~x Fig. 8. Reassoeiation kinetics of nuclear polyhedrosis virus D N A as a function of Cot

Figure 8 shows the kinetics of N P V DNA renaturation. The extent of renaturation of nonrepeated DNA sequences was predicted b y the formula of BRITTEN and K e d g e (1) : C 1 Co -- 1 + K2Cot where: C : concentration of unpaired DNA strands (reel. nucleotides]L); Co = total concentration of DNA (reel. nuclcotides/L); Cot = concentration of DNA × time (mol.×sec./L); K2 ~ reaction rate constant (L/mol. ×see.); 1 C 1 ~-: Cotl/2 (the value of Cot when Coo~ 2)" 2

The experimental points were, within the limits of the technique, found on a straight line (C0/C against Cot) indicating the absence of repeated D N A sequences. Assuming a linear relationship between Co%/2 and genetic complexity (1, 10) and a Cotl/2 value of 9.8 for E. cell which has a genome size of 2.8 × 109 (2), we estimate the genome size of N P V to be about 77 × 106 (Cotl/2 = 0.27). The mol. weight of the N P V genome was corrected to take account of the fact t h a t D N A renaturation is retarded with increasing GC content (18). Such a correction produces a Co%/2 value for N P V of 0.33 and a N P V genome size of about 92 × 106. Discussion

GmL NPV particles non-occluded into polyhedra are labile (12) and difficul~ to purify without loss of infectivity. D N A isolated from virus prepared on sucrose gradients contains non-labelled UV-adsorbing material. CsC1 density gradient

Macromoleeular Structure of Nuclear Polyhedrosis Virus Genome

341

centrifugation has been used to prepare NPVs of Trichoplu~ia ni and Spodoptera ]rugiperda from polyhedra (20). Although non-occluded virus particles are degraded on CsC1 purification, it is possible to isolate infectious DNA from them. This purification procedure is useful when purity of DNA is more important than the production of undegraded DNA molecules. Storage conditions of the viral preparations were shown to influence DNA structure. Supercoflcd DNA was obtained from ]yophilized virus (non-occluded NPV particles suspended in water) unlike S V ~ E R S and A~D~RSO.~ (20) who failed to isolate supercoiled DNA from lyophilized virus. Nicking of DNA molecules appeared not to be caused by lyophi]ization, but as a result of other factors. Sucrose gradient centrifugation in high ionic strength neutral solutions have previously been used to fractionate the different forms of PM2 DNA (13) and the intraeellular forms of Epstein-Barr virus (l 1). Using this method, we were able to divide covalently closed DNA molecules from open circular and linear ones, and to confirm heterogeneity in supercoiled molecules of the same size. I t was difficult to differentiate supercoiled DNA from circular DNA molecules (Fig. 3 A, B, C), and only in the presence of E t h B r molecules did take up a supercoiled configuration (Fig. 3D). I t is not clear why covalently closed DNA molecules have variable densities in high ionic strength neutral gradients. DNA-associated proteins have been reported to increase DNA sedimentation coefficients (4). We cannot exclude the possibility that there are some pronase-resistant DNA-protein complexes in our preparations (22) althoug pronase treatment did not change the sedimentation and biological properties of our preparations. Heterogeneity in the superhelical densities of superhelical DNA molecules has been described for polyoma virus, SV40 and PM2 DNAs (5, 14, 16). Similarly, covalently closed molecules from the two sucrose density fractions in our experiments might also differ in their superhetix denMty (23). I t was difficult to determine the superhelix density of covalently dosed NPV DNA molecules because of their low concentrations and lability (17). Electron microscopic data on molecular size, and biological activity tests confirmed that only covMently closed molecules or mixtures of open circular and linear molecules of contour length about 48--50 ~ (about 100 × 106) were infectious. I t is interesting to note that in the DNA sedimentation profiles (Fig. t A, 13) peak I I I infectivity is found in more dense fractions than the radioactivity. Circular DNA molecules usually have higher sedimentation coefficients than linear molecules of the same size (21). These results suggest the infectivity of only circular DNA molecules and non-infectivity of linear molecules of the same size. Non-infectious linear mMecules of contour length up to 20--25 ~z appeared after viral DNA degradation. The absence of infectivity in these molecules m a y be due to breaks in the helices of circular molecules, as such breaks have been shown to inactivate some small viral circular DNAs (3). Our reassociation kinetics experiments have demonstrated unique nucleotide sequence as long as 90 × 106. Infectious circular DNA molecules are of this size. Preliminary data on the genome size determined from the molecular weight of restriction enzyme fragments also indicate a genome size of about 90 × 106.

342

L.I. ST~OKOVS~ASrA

et ai. :

However, recent reports indicate, b y using kinetic reassociation analysis, t h a t the genome size for NPVs of several S~)odop~era species range from 62 to 82>

Macromolecular structure of nuclear polyhedrosis virus genome.

Archives of Virology Archives of Virology 59, 331--343 (1979) © by Spmnger-Vertag 1979 Macromoleeular Structure of Nuclear Polyhedrosis Virus Genom...
1MB Sizes 0 Downloads 0 Views