The infectivity of nuclear polyhedrosis virus DNA

Ann. Virol. (Inst. Pasteur) 1981, 132 tl, 247-259

THE OF

NUCLEAR

INFECTIVITY

POLYHEDROSIS

VIRUS

DNA

by D. C. Kelly and X. Wang (*) N. E. R. C., Institute o/ Virology, Mansfield Road, Ox]ord (UK)

SUMMARY Trichoplusia ni multiply-enveloped nuclear polyhedrosis virus DNA is infectious provided it is circular and double-stranded. This has been shown by comparing linear, nicked circular (nc) and covalently closed (cc) DNA, with single-stranded derivatives, and by comparing naturally occurring forms with forms generated enzymatically with ligase and S1 nuclease, cc-DNA has a low superhelical density and is as infectious as nc-DNA. The infectivity of the DNA is enhanced by basic proteins such as protamine and the major basic nucleocapsid protein of Heliothis zea baculovirus. The host range of the infectious DNA paralleled t h a t of live virus and was restricted to lepidopteran cell lines.

KEY-WORDS: Nuclear polyhedrosis virus, DNA; Infectivity.

Nuclear polyhedrosis virus (NPV) DNA is infectious [6, 8, 11]. The viral genome is covalently closed, has a superhelical density approaching zero and a size of about 80 • 106 [41, 34, 5, 9]. Both covalently closed (cc) and nicked circular (nc) DNA are infectious, and Burand et al. [8] have reported that cc-DNA is four times as infectious as nc-DNA. Linear duplex DNA created by controlled shearing of circular DNA has been shown to be non-infectious [6]. This study had a number of objectives. Firstly to investigate the nature of the infectivity of nc- and cc-DNA using a variety of DNAmodifying enzymes and physical treatments. Secondly to investigate the in vitro host range of the DNA; this was done because it was claimed by Burand el al. [8] t h a t the in vitro host range of infectious baculovirus DNA Manuserit re~u le 26 mai 1981, acceptd le 4 juin 1981. (*) On sabbatical leave from the Institute of Entomology, Zhongshan University, Guangzhou (China).

248

D. C. K E L L Y AND X. WANG

is i d e n t i c a l to t h a t of live v i r u s a l t h o u g h o n l y one of t h e five i n v e r t e b r a t e cell lines used was n o n - p e r m i s s i v e . T h i r d l y , a basic p r o t e i n is i n t i m a t e l y a s s o c i a t e d w i t h t h e D N A e x t r u d e d f r o m n u c l e o c a p s i d s [42, 7] a n d we wished to d e t e r m i n e t h e effect of this p r o t e i n h a s on t h e i n f e c t i v i t y of b a e u l o v i r u s D N A . T h e i n t e r a c t i o n of Trichoplusia ni m u l t i p l y - e n v e l o p e d N P V w i t h Spodoplera frugiperda cells was u s e d as t h e m o d e l s y s t e m in this s t u d y . METHODS

Virus production. The following baeuloviruses were routinely produced in their natural insect hosts: multiply-enveloped NPV from T. hi, S. [rugiperda, S. liltoralis, S. lilura and Mamestra brassicae, a singly-enveloped NPV from Heliothis armigera and granulosis viruses (GV) from S. [rugiperda and S. litloralis. As previously described [4, 26, 27]. T. ni NPV was also produced in S. [rugiperda cells [6].

Cell culture. The following cell lines were used: S. [rugiperda (1PBL-2 [43]), Bombgx mori [16], M. brassicae [29], T. ni (TN-368 [21]), Drosophila melanogasler [36], Lymantria dispar [32], Aedes albopiclus [39], Carpocapsa pomonella [22], Veto [46], baby hamster kidney 21 [28], primary chick embryo fibroblasts, bluegill fry (BF2 [47]), fat head minnow (FHM [19]), rainbow trout gonad (RTG2 [45]), Chinook salmon embryo (CHSE [14]) and Xenopus laevis (XTC2 [31]). The fish, amphibian, avian and mammalian cells were grown in Eagle's minimal essential medium supplemented with 10 ~o calf serum as previously described [13]. The fish cells were grown at 21 ~ C, the amphibian cells at 28 ~ C and the other vertebrate cells at 37 ~ C. The invertebrate cells were grown at 28 ~ C and were grown in TC100 medium [15] with the exception of the D. melanogasler cells which were grown in Schneider's medium [35].

DNA purification. DNA was purified from purified virus particles as described by Archard and Mackett [1]. Covalently closed DNA and relaxed DNA was resolved on ethidium bromide-caesium chloride gradients, and the DNA bands recovered from these gradients were t r e a t e d with butanol to remove the ethidium bromide prior to overnight dialysis in three changes of sterile water. DNA amounts were estimated from the OD at 260 nm (50 ~g/inl = OD 1 in a 1 cm path). DNA at a 260-280 nm ratio of at least 1.75 was used.

In/ectious DNA assay. The assay was based on the method described by Graham et al. [18], using the conditions previously determined [6]. DNA derived from insect grown virus was routinely used. DNA was diluted in Hepes buffer p H 7.0 (0.02 M Hepes, glucose 0.01 M, Na2HPO4 0.001 M, KC1 0.005 M and NaC1 0.14 M). One hundred ~1 of salmon sperm DNA (100 ~g/ml) and 65 ~1 of 2 M CaC12 were added to 900 ~1 of viral DNA, and the mixture was incubated at room temperature for 15 min cc = covalently closed. GV = granulosis virus.

I nc : nicked circular. I NPV = nuclear polyhedrosis virus.

I N F E C T I V I T Y OF NPV DNA

249

before adding to cells. Routinely S. [rugiperda cells (10 s cells/3.5 cm Petri dish) incubated at 23 ~ C for 16 h were used. The medium TC100 was removed, and 1 ml of CaC12-precipitated DNA was added to the cells. Absorbtion took place for 1 h at 28 o C and the inoculum was removed and replaced with 1 ml of TC100 for 4 h. The TC100 was removed and replaced with a 2 ml overlay comprising equal amounts of 3 % agarose (LGT agarose, Miles Laboratories Ltd., Stoke Poges, UK) in water and TC100, and this was supplemented on solidification with 1 ml of TC100. The dishes were incubated at 28 ~ C for 72 h, then 1 ml of 0 . 1 % (w/v) neutral red in PBS was added, and the dishes were incubated for another 8 h. T h e liquid overlay was decanted and the plaques were read 16 h later after incubation at room temperature.

Plaque assay o[ baculoviruses. This was done essentially as described above in the infectious DNA assay. One hundred ~l of virus were allowed to absorb to the cell monolayer. Routinely S. [rugiperda cells were used as before. The inoeulum was removed, the agarose overlay was added, and the procedure was thereafter as for the infectious DNA assay. Elhidium bromide-caesium chloride D N A centri/ugation. The procedure of Radloff el aI. ]33] as described by Payne [30] was used. Briefly 1 ml baculovirus lysed in sareosine was layered on a 8 ml 54 ~ (w/w) CsC1 in 0.01 M-Tris-HC1 pH 8.0 containing 10 mM EDTA and 100 ~g/ml ethidium bromide; gradients were formed by centrifuging for 24 to 48 h at 60,000 g at 25 ~ C (MSE Superspeed 60, 6 • 15 ml, swing-out rotor); 0.1 ml fractions were collected.

Electron microscopg o/ DNA. It was performed as described by Bud and Kelly [5]. Agarose-geI eleclrophoresis o[ DNA. Intact and restriction enzyme-digested DNA were resolved on 0.3 % and 1.0 % gels as previously described [27]. The number of superhelical turns in polyoma DNA was determined by the two gel systems described b y Shure and Virograd [38]. Treatment of D N A with enzymes. Six ~1 of enzyme were added to 140 ~1 of 2 • buffer and 135 ~l of DNA (2 ~g/ml water). The individual treatments were for 60 min at 37 ~ C. Subsequently the samples were made up to 2.7 ml with Hepes buffer; 300 ~l of salmon sperm DNA (100 ~g/ml) and 195 ~l of CaCl~ 2M were added. The infectious DNA was then assayed in 1 ml amounts as previously described. The following enzymes were used: S1 nuclease, DNase 1, RNase ex bovine pancreas (all Sigma Chemical Co. Ltd. London), proteinase K (Boehringer Corporation Ltd. London), the restriction enzymes SmaI and XhoI, T4-DNA ligase, and DNA-relaxing enzyme (all Uniscience Ltd., Cambridge). The following amounts of enzyme and buffer were used: S1 nuclease (100 units) and a buffer comprising sodium acetate 30 raM, p H 4.7, NaC1 50 mM, Z n S Q 1 mM and 5 ~o glycerol; DNase (2 ~g) and Hepes supplemented with MgC12 6 raM; RNase (2 mg) and Hepes; proteinase K (2 ~g) and Hepes supplemented with MgCl~ 30 raM; Xhol (3 units) and 8 mM Tris-HC1 (pH 7.4), NaC1 150 mM, MgCI~ 6 mM and mercaptoethanol 6 mM; Sinai (3 units) and 15 mM TrisHC1 (pH 8.0), MgCl~ 6 mM and KCl 15 mM; T4-DNA ligase (10 units) and 66 mM Tris-HC1 (pH 7.5), MgCl~ 6.6 raM, ATP 66 mM and dithiothreitol 10 mM; DNArelaxing enzyme (5 units) and 20 mM Tris-HC1 (pH 7.8), MgC12 2 mM and 2-mercaptoethanol 7 mM. Ann. V~ro'~. (Inst. Past.), 13~ E,

n ~ 2, 1981.

17

250

D.C.

KELLY AND X. WANG

Trealment o[ DNA with baculovirus basic protein and protamines. The basic protein (molecular weight 13,700 daltons) was purified from Heliothis zea singly enveloped NPV [27] by acid extraction (D. C. Kelly, D. A. Brown, M. D. Ayres, C. J. Allen and I. O. Walker, manuscript in preparation). Protamine sulphate was obtained from the Sigma Chemical Co. London Ltd. These proteins were allowed to interact with baculovirus DNA at 37 ~ C for 60 rain prior to assay. Ten ~1 of either protein (containing 5 to 20 ~g/ml) were added to 135 ~1 of DNA (2 ~g/ml). After incubation 2.565 ml of Hepes buffer were added, and the DNA infectivity was then assessed.

Preparation o[ single-stranded circular T. ni-NPV DNA. Alkaline sucrose gradients capable of resolving linear and circular singlestranded DNA were run as described by Kelly and Avery [25].

RESULTS

The nalure of T. n i - N P V D N A . We have previously shown t h a t the viral DNA is a cc double-stranded DNA molecule with a MW of about 8.0 • 107 daltons [6]. R e c e n t l y R e v e t and Guelpa [34] d e m o n s t r a t e d t h a t the ec-DNA of the singly-enveloped baculovirus from a dipteran Tipula paludosa had a low superhelical density. Since this is an i m p o r t a n t characteristic not determined for other baeuloviruses, and the T. ni NPV by contrast is a multiply-enveloped lepidopteran baeulovirus, we have examined the superhelieal density of T. ni-NPV ec-DNA by two s t a n d a r d methods: b u o y a n t density determinations in the presence of an intercalating dye and electron microscopy [2].

FIG. 1. - - Separation of nc- and c c - D N A (upper and lower bands respectively) o[ T. ni N P V and baeleriophage P M 2 ( B ) on ethidium bromide-caesium chloride gradients.

(A)

INFECTIVITY OF NPV DNA

251

Figure 1 shows the resolution of cc-DNA and relaxed D N A of T. n i - N P V D N A and bacteriophage PM2 (used as a standard) on ethidium bromidecaesium chloride gradients. The N P V cc-DNA has a higher b u o y a n t density t h a n PM2 cc-DNA. We use the formula derived b y Gray et al. [20] modified b y t l e v e t and Guelpa [34]: r1 1 ~o = 0.80 ] ~ ( r A r ) / ( r 5 } ) + | [.1~ J

0.134

where ~o is the n u m b e r of superhelical turns per Watson-Crick duplex turn; f is the distance from the centre of rotation to the average location between a closed-open cognate pair; Ar is the separation between corresponding band centres; the superscript ( § refers to PM2 DNA; and f+ accounts for any difference in the base composition of the unknown closedrelaxed pair and t h a t of PM2 D N A [10]. Taking the b u o y a n t CsC1 density of T. n i - N P V D N A as 1.701 g/ml (D. C. Kelly, unpublished observations) and t h a t of PM2 D N A as 1.703 g/ml, the calcutated figure for (f+) is 1.006. Measurements of the banding positions of cc-DNA and relaxed D N A in figure 1 show t h a t the average value of Ar/Ar+ is 1.70 • 0.04 (r+ = 11.789 cm and ~ = 11.929 cm), and so it follows t h a t ~o for T. n i - N P V cc-DNA is 0.001 ~-0.0001. A comparison of cc-DNA from cell grown virus and insect grown virus showed t h a t t h e y were identical in b u o y a n t density and so superhelical density (data not shown). Electron microscopy of T. n i - N P V D N A produced b y a proteinase K-phenol extraction showed evidence of cc-DNA. A b o u t 5 % of molecules were twisted (fig. 1 b in [5] shows such structures). Counting of cross-overs in 15 molecules showed t h a t an average of 90 • 8 cross-overs per molecule occurred. The average length of (< relaxed )~ circular D N A of T. ni N P V is 38.36 ~m, and since the molar linear density was calculated to be 2.075 • l@/~,m [6], there are 2.346 • 0.208 cross-overs per ~m, and so a superhelical density of 0.007 • 0.001. It must be stressed t h a t this is not an absolute value because variable surface spreading forces induce writhing which is intereonvertible with twisting on the application of torque [2, 45], and it merely confirms the low superhelical density of T. n i - N P V DNA. A t t e m p t s to use the band counting methods of Shure and Vinograd [38] and Keller [24] which use relaxing enzymes failed to resolve bands of differing superhelical density (data not shown).

Fundamental properties infectious T. n i - N P V D N A . D N A derived from virus particles purified from insect grown polyhedra, were used for convenience since it is easier to obtain large quantities of such material compared with cell culture grown virus. A comparison of cc- and nc-DNA from insect and cell grown virus is shown in table I. No significant differences in infectivity was detected between the four sources. The p. f. u./~g ration was routinely in the 300 to 500 ratio although exceptionally values of up to 2,000 were obtained. T r e a t m e n t with dimethylsulfoxide, which enhances the infectivity of

252

D. C. KELLY AND X. WANG

some viral DNA in certain vertebrate cells [40] failed to significantly increase the DNA infectivity. T r e a t m e n t of the DNA with DNase and the restriction enzymes XhoI and Sinai, destroyed infectivity whereas RNase and proteinase K had virtually no effect (table II). Single-stranded circular and linear DNA obtained from alkaline sucrose gradients were not infectious. TABLE I. - - Infectivity of (( T. ni ))-NPV DNA obtained from insect and ceil grown virus (in p. f. u./~g DNA). Source

cc-DNA

nc-DNA

Insect grown virus Cell g r o w n v i r u s

450 • 10 444 • 15

475 ~_ 11 377 • 10

TABLE II. --- Effect of various enzymes on infectivity of (( T. ni )~-NPV DNA (in p. f. u./~g DNA). Enzyme

cc-DNA

nc-DNA

DNase RNase Proteinase K E n d o IR.XhoI Endo H.SmaI Control

0 560 • 25 320 • 19 0 0 640 • 20

0 407 • 17 300 • 17 0 0 425 • 23

Effect of D N A modifging enzgmes on T. n i - N P V D N A infectivitg. S1 nuclease, a single-strand specific DNase can nick supercoiled DNA to produce nc-DNA which in t u r n is converted to linear DNA [3]. As shown in table III, S1 nuclease destroyed the infectivity of both cc- and nc-DNA. T4-DNA ligase will convert nc-DNA to cc-DNA; t r e a t m e n t of both ccand nc-baculovirus DNA failed to enhance DNA infectivity (table III). T r e a t m e n t with DNA-relaxing enzyme (which progressively unwinds superhelical DNA) considerably enhanced the infectivity of both nc- and cc-DNA. TABLE III. - - Effect of DNA-modifying enzymes on infectivity of (( T. ni )~-NPV DNA (in p. f. u./~g DNA). Enzyme

cc-DNA

nc-DNA

$1 n n c l e a s e T 4 - D N A ligase DNA-relaxing enzyme Control

0 136 • 8 520 • 32 130 • 6

0 167 • 7 656 • 85 175 • 4

INFECTIVITY OF NPV DNA

253

Effect of heat and alkali trealmenI on D N A infeclivitg. cc-DNA generally is more resistant to extremes of acid and heat than nc-DNA [2]. Preliminary work showed t h a t on heating polyhedra in water at t e m p e r a t u r e s of 600 C or higher for 30 rain no cc-DNA was detected on ethidium bromide-CsC1 gradients. The nc-DNA and linear D N A so obtained were assayed and it was found t h a t D N A obtained from polyhedra heated at 60 ~ C had considerably reduced infectivity (ca. 4 %), and D N A obtained from polyhedra treated at higher t e m p e r a t u r e s was completely uninfectious (table IV). Analysis of nc-DNA preparation on 0.3 % agarose gels showed t h a t most of t h e D N A obtained at these t e m p e r a t u r e s was linear (data not shown). TABLE IV. - - Infectivity of << T. ni ~>-NPV D N A obtained thermally inactivated polyhedra (in p. f. u./~g DNA). Source Pelyhedra, Polyhedra, Polyhedra, Polyhedra, Polyhedra,

9 0 ~ C, 3 0 m i n 8 0 ~ C, 3 0 m i n 70 ~ C, 3 0 m i n 60 ~ C, 3 0 m i n room temp.

(*) N o c e - D N A

cc-DNA

ne-DNA

(*) ---600 i 20

0 0 0 244 i 33 666 i 33

recovered.

Denaturation of nc- and ce-DNA in Hepes buffer before assay b y heating at 96 ~ C for 10 min considerably reduced the infectivity of both ec- and nc-DNA, the nc-DNA infectivity being reduced to a greater e x t e n t (table V). Alkali denaturation b y treating viral D N A with 0.1 N N a O H for 15 rain at 24 ~ C followed b y neutralisation, destroyed the infecti~dty of both t y p e s of D N A (table V). TABLE V. Effect of heat and alkali treatment on << T. ni ~>-NPV DNA infectivity (in p. f. u./~g DNA). -

-

Treatment

cc-DNA

A l k a l i , 0 . 1 N N a O H , 15 r a i n t h e n n e u t r a l i s a t i o n H e a t , 96 ~ C, 1 0 m i n t h e n c o o l i n g o n ice Control

0 4 6 5- 6 988 • 66

nc-DNA 0 54 :k 8 1 , 9 2 2 :k 8 3

Effect of basic proteins on D N A infectivitg. The infectivity of both cc- and nc-DNA was enhanced b y addition of b o t h the basic proteins from H. zea (a major nucleocapsid DNA-binding protein) and protamine (taMe VI). Complete nueleocapsids, which are

254

D.C.

KELLY AND X. WANG

TABLE V I . - - Effect on basic proteins on i n f e c t i v i t y of a T. ni >>-NPV D N A (in p. f. u . / ~ g D N A ) .

Protein Control P r o t a m i n e (1) H. zea nucleocapsid basic protein (2) T. ni nucleocapsid in toto T. ni nucleocapsid (*) in toto

ec-DNA 400 674 1,600 720

~_ • i •

28 35 105 32

ne-DNA 444 • 23 720 • 20 1,850 • 47

0

(*) A s s a y e d b y direct p l a q u e assay. (i) 0.75 ag p r o t a m i n e / ~ g D N A . (2) 0.75 [xg n u c l e o e a p s i d basic protein/iag D N A .

not infectious b y conventional plaque assay, were infectious when processed through a DNA-infectivity assay and the infectivity was approximately equal to t h a t of naked DNA.

Host cell range

of T.

ni-NPV DNA.

The interactions of infectious D N A and live virus with a variety of cell lines assayed b y DNA-infectivity assay and plaque assay are shown in table VII. Infectivity assessed in both systems was detected in just S. frugiperda and T. ni cell lines.

TABLE V I I . - - (( In vitro >> host range of i n f e c t i o u s ~ T. ni >>-NPV D N A .

Positive: Negative:

S. [rugiperda, T. ni L g m a n t r i a dispar (*), Mamestra brassicae (*), Bomb!Ix mori, Heliothis zea, Drosophila melanogasler , A e d e s albopictus, Carpocapsa pomonella, X e n o p u s laevis F a t h e a d m i n n o w , bluegill fry, r a i n b o w t r o u t Vero, B H K 21/13 Chick e m b r y o fibrohlasts

gonad, Chinook s a h n o n e m b r y o

(*) T h e s e cell lines s u p p o r t t h e replication of live v i r u s inefficiently. O t h e r n e g a t i v e cell lines do n o t s u p p o r t t h e replication of live virus.

Susceptibility of S. frugiperda and T. ni cells to other baculovirus. DNA, from S. frugiperda N P V and GV, S. litura NPV, S. liltoralis N P V and GV, M. brassicae N P V and H. armigera NPV, both ne and ee failed to initiate plaques in S. frigiperda and T. ni cells.

INFECTIVITY OF NPV DNA

255

DISCUSSION Our studies extend the observations previously made on infectious baculovirus DNA. The DNA is infectious provided it is double-stranded and circular. We have found no evidence for greater infectivity of cc-DNA and ne-DNA. This contrasts with observations made on papovavirus DNA [19.] where the cc-DNA is four times as infectious as nc-DNA, and this probably reflects the marked difference in superhelical density of the DNA contained by papovaviruses and baculoviruses. The observation that there is little difference in infectivity between the two forms of baculovirus DNA is important. Baculoviruses can be used as biological insecticides and, currently, considerable effort is being made to formulate virus preparations of high virulence in both insects and cell culture. Routinely, yields of more than 30 ~ cc-DNA are rarely obtained from viral DNA preparations (which normally are greater than 95 % circular), and the cc-DNA gradually relaxes to nc-DNA on storage (D. C. Kelly, unpublished observations). It is unlikely therefore, t h a t efforts to produce and store virus containing a greater proportion of ec-DNA will provide virus of enhanced virulence. The demonstration that T. ui DNA has a low superhelical density, irrespective of whether it was produced in insects or cell culture, confirms the observation of Revet and Guelpa [34]. A preliminary screen of other baculoviruses indicates t h a t this is a general phenomenon (D. A. Brown and D. C. Kelly, unpublished observations), and this is probably related to the overall strategy of replication and packaging of the DNA into rod-shaped nucleocapsids. Linear double-stranded DNA, DNA specifically fragmented with restriction enzymes, and single-stranded DNA was not infectious. The observation that single-stranded circular DNA was not infectious is interesting since, for example, herpes virus linear single-stranded DNA is infectious [37]. It is possible that single-stranded DNA is susceptible to nuclease digestion and the circular DNA is efficiently cleaved before a complementary strand is synthesized, so producing non-infectious linear double-stranded molecules. Both the ce- and nc-DNA was rendered non-infectious by digestion with strand specific S1 nuclease. Monitoring on gels showed t h a t the enzyme converted both forms to full length linear molecules, and this provides additional evidence t h a t linear DNA is not infectious. S1 nuclease is able to cleave superhelical DNA because regions of unpaired or weakly hydrogen banded regions are present in the molecule [3]. Ligation of ncDNA to produce cc-DNA was efficiently accomplished with T4-DNA ligase, and manufacture of ec-DNA in this way to produce cc-DNA of zero superhelical density failed to enhance the infectivity of the DNA. Attempts to increase the superhelical density of the cc-DNA by ligating in the presence of increasing amounts of ethidium bromide also failed to enhance DNA infectivity (D. C. Kelly and X. Wang, unpublished observa-

256

D. C. KELLY AND X. WANG

tions), but since insufficient cc-DNA to monitor superhelicity was produced, one cannot yet correlate infectivity with superhelical density. The converse experiment of reducing the superhelieal density in naturally occurring baculovirus cc-DNA by using DNA-relaxing enzyme, a topo-isomerase, enhanced its infectivity; the control experiment with ne-DNA also enhanced DNA infectivity. Although we have not directly shown this, it is probable t h a t t h e effect is due not to the modification of superhelieal density of the DNA but to the general effect of a DNA-binding protein which effectively protects the D N A from nuclease digestion and m a y orientate the genome for correct interaction with transcribing and replicating enzymes. T r e a t m e n t of both he- and cc-DNA with basic DNA-binding proteins - - t h e basic protein from H. zea N P V nucleocapsids or protamines from salmon testes - enhanced DNA infectivity considerably. The optimal a m o u n t of the basic protein required to enhance D N A infectivity was equivalent w/w to t h a t found naturally in viral nucleocapsids. Nucleocapsids which are not naturally infectious (presumably because t h e y lack the receptors present in the viral envelope) became infectious when assayed in an DNA-infectivity assay. The susceptibility of viral DNA infectivity to heat and alkali was predictable. The infectivity was destroyed by extremely alkaline pH in the case of both nc- and cc-DNA. This is compatible with the observation t h a t single-stranded DNA is not infectious. The effect of heat showed t h a t cc-DNA was more thermoresistant t h a n nc-DNA although it was surprising some infectivity was retained with nc-DNA. Kinetic analysis of the thermal inactivation should highlight the differential inactivation. Thermal inactivation of DNA in polyhedra showed t h a t DNA was more susceptible to inactivation in situ t h a n when released into an aqueous environment. This was probably because within polyhedra constraint on viral DNA caused double-stranded breaks to occur so creating noninfectious linear DNA. We plan to further investigate this phenomenon with respect to the lethal dose 50 of the virus since heat inactivation of polyhedra in the field, particularly when absorbed to leaf surfaces, is an i m p o r t a n t factor when using the virus as a biological insecticide. The host cell range of infectious DNA was studied to evaluate whether, in the absence of specific virus receptors in the envelope, one could extend the range of the virus. As reported b y B u r a n d et al. [8] the host cell range of the DNA matches t h a t of live virus, at least if ability to produce plaques in a monolayer is a criterion of infectivity. Using this criterion, the DNA was unable to replicate in m a m m a l i a n , avian, piseine and amphibian cells, together with a n u m b e r of invertebrate cells. The DNA was infectious for just S. frugiperda and T. ni cells despite the fact t h a t the virus infects, albeit inefficiently, M. brassicae and L. dispar cells (T. Lescott, X. W a n g and D. C. Kelly, unpublished observations) although the virus does not plaque in these cells under our assay conditions. We were also unable to initiate infection in S. frugiperda and T. ni cells with GV and N P V which normally fail to initiate infection in these cells (although M. brassicae N P V replicates ineffmiently in S. frugiperda cells

INFECTIVITY OF NPV DNA

257

(D. C. Kelly, unpublished observations)). This demonstrates inability for a baculovirus to initiate infection is not due solely to lack of cell receptors for virus proteins involved in a t t a c h m e n t .

RESUMe: P O U V O I R I N F E C T A N T DE

L'ADN

D U VIRUS DE LA POLYYIEDROSE N U C L E A I R E

L ' A D N du virus multi-envelopp6 de la polyh6drose nucl6aire de Trichoplusia ni est infectieux ~ condition qu'il soit sous forme circulaire et bicat6naire. On a montr6 cela en c o m p a r a n t le caract~re infectieux d'une part des formes lin6aire, circulaire relax~e (nc) et superh61ico~dale (cc) aux d6riv6s monocat6naires et, d'autre part, des formes naturelles ~ celles obtenues ~ l'aide d'enzymes (ligase et nucl6ase S1). L ' A D N superh61icoYdal a une faible densit6 de supertours et il est aussi infectieux que l'est la forme circulaire relax~e. Le pouvoir infectant de I'ADN est augments par adjonction de prot6ines basiques telles la protamine ou la prot6ine basique majeure de la nucl~ocapside du baculovirus de Heliolhis zea. Le spectre d'h6tes de I'ADN infectieux est semblable a celui du virion et se limite aux lign6es cellulaires de 16pidopt~res. MOTS-CLI~S : Virus de la polyh~drose nucl6aire, A D N ;

InfectivitY.

REFERENCES [1] ARCHARD, L. C. & MACKETT, M., Restriction endonuclease analysis of red cowpox virus and its white pock variant. J. gen. Virol., i979, 45, 51-63. [2] BAUER, W. R., Structure and reactions of closed duplex DNA. Ann. Reg. Biophgs. Bioengineer., 1978, 7, 287-313. [3] BEARD, P., MORROW, 3. F. & BERG, P., Cleavage of circular, superhelical simian virus 40 DNA to a linear duplex by $1 nuclease, d. Virol., 1973, 12, 1303-1313. [4] BROWN, D. A., BUD, H. M. & KELLY, D. C., Biophysical properties of the structural components of a granulosis virus isolated from the cabbage white butterfly (Pieris brassicae). Virology, 1977, 81, 317-327. [5] BUD, H. M. & KELLY, D. C., The DNA contained by nuclear polyhedrosis viruses isolated from four Spodoptera spp. (Lepidoptera: Noctuidae); genome size and configuration assessed by electron microscopy. J. gen. Virol., 1977, 37, 135-143. [6] BUD, H. M. & KELLY, D. C., Nuclear polyhedrosis virus DNA is infectious. Microbiologica, 1980, 3, 103-108. [7] BUD, H. M. & KELLY, D. C., An electron microscope study of partially lysed baculovirus nucleocapsids: the intranucleocapsid packaging of viral DNA. d. Ultrastruct. Res., 1981, 1, 73, 361-368. [8] BURAND, J. P., SUMMERS, M. D. & SMITm G. E., Transfection with baculovirus DNA. Virology, 1980, 101, 286-290.

258

D.C.

K E L L Y AND X. WANG

[9] BURGESS, S., Molecular weights of lepidopteran baculovirus DNAs: derivation by electron microscopy. J. gen. Virol., 1977, 37, 501-510. [10] BURKE, R. L. ~r BAUER, }V. R., Measurement of superhelix densities in buoyant dye/CsC1. Proc. nat. Aead. Sci. (Wash.), 1977, 152, 291-292. [11] CARSTENS, E. B., TJIA, S. T. ~r DOEFLER, W., Infectious DNA from Aulographa cali[oruica nuclear polyhedrosis virus. Virology, 1980, 101,311-314. [12] DULnECCO, lq. & VOGT, M., Evidence for a ring structure of polyoma virus DNA. Proe. nat. Acad. Sci. (Wash.), 1963, 50, 236-241. [13] ELHOTT, R. M., ARNOLD, M. K. & KELLY, D. C., The replication of frog virus 3 in an amphibian cell line (XTC-2) derived from Xenopus laevis. J. yen. Virol., 1979, 44, 89-98. [14] FRYER, J. L., YUSHA, A. & PILCttER, K. S., The in vitro cultivation of tissues and cells of Pacific salmon and steelhead trout. Ann. N. Y. Aead. Sci., 1965, 126, 566-586. [15] GARmNER, G. R. & STOCKDALE,H., Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Invertebr. Path., 1975, 25, 303-370. [16] GRACE, T. D. C., Establishment of a line of cells from the silkworm Bombgx mori. Nature (Lond.), 1967, 216, 613-614. [17] GRAHAM,F. L. & VAN DER EB, A. J., A new technique for the assay of infectivity of adenovirus 5 DNA. Virology, 1973, 52, 456-467. [18] GRAHAM, F. L., VELDHUmEN, G. & WILgIE, N. M., Infectious herpes DNA. Nature New Biol. (Loud.), 1973, 245, 265-266. [19] GRAVELL,M. & MALSBERGER, R. G., A permanent cell line from the fathead minnow (Pimephabs promelas). Ann. N. Y. Acad. Sei., 1965, 126, 555-565. [20] GRAY, H. B., UPHOLT, W. B. & VINOGRAB, J., A buoyant method for the determination of the superhelix density of closed circular DNA. J. tool. Biol., 1971, 62, 1-19. [21] H:NK, W. F., Established insect cell line from the cabbage looper, Trichoplusia hi. Nature (Lond.), 1970, 226, 466-467. [22] HINK, U. F. & ELLIS, B. F., Establishment of two new cell lines (CP-1268 and CP-129) from the codling moth Carpsocapsa pomonella. Curt. Top. Microbiol. Immunol., 1971, 55, 19-28. [23] HINK, W. F. & IGNOFFO, C. M., Establishment of a new cell line (IMC-HZ-1) from ovaries of cotton bollworm moths Heliothis zea (Boddie). Exp. Cell Rcs., 197l, 60, 307-309. [24] KELLER, W., Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. Proc. nat. Aead. Sci. (Wash.), 1975, 72, 4876-4880. [25] KELLY, D. C. & AVERY, B. J., Frog virus 3 deoxyribonucleic acid. J. gen. Virol., 1974, 24, 339-348. [26] KELLY, D. C. ~5 BROWN, D. A., Biochemical and biophysical properties of a 31amestra brassicae multiply enveloped nuclear polyhedrosis virus. Arch. Virol., 1980, 66, 133-141. [27] KELLY, D. C., BROWN,D. A., F{OBERTSON,J. S. & HARRAP, K. A., Biochemical, biophysical and serological properties of two singly enveloped nuclear polyhedrosis viruses from Heliolhis zea and Heliothis armigera. ~lierobiologica, 1980, 3, 319-331. [28] MACPHERSON, I. A. & STOKER, M. G. P., Polyoma transformation of hamster cells clones, an investigation of the genetic factors affecting cell competence. Virology, 1962, 16, 147-152. 129] MILTE~BURGER, H. G., DAWD, P., MAHR, U. & ZIPP, W., 15bet die Estellung yon Lepidopteran-Daverzellinien und die in vitro replication yon insektenpathogen viren. - - I. Mamestra brassicae Zellinien und NPV replikation. Zeutblat any Entomol., 1977, 82, 306-323. [30] PAYNE, C. C., The isolation and characterisation of a virus from Orgctes rhinoceros. J. gen. Virol., 1974, 25, 105-116.

INFECTIVITY OF NPV DNA

259

[31] PUDNEY, M., VARMA, M. G. R. & LEAKE, C. J., Establishment of a cell line [32] [33]

[34]

[35] [36] [37] [38] [39] [40] [41] [42] [43] [44]

[45] [46] [47]

from the South Africa clawed toad, Xeuopus laevis. Experientia (Basel), 1973, 29, fl66-467. QuroT, J. M., Etablissement d'une lignSe cellulaire (SCLd135) h partir d'ovaires du L6pidopt~re Lgmantria dispar. C. R. Acad. Sci. (Paris) (S6r. D), 1976, 282, 465-467. I~ADLOFF, R., BAUER, W. & VINOGARD, J., A dye buoyant density method for the detection and isolation of closed circular duplex DNA, the closed circular DNA in HeLa cells. Proe. t~at. Acad. Sci. (Wash.), 1967, 57, 1514-1521. REVET, B. M. J. & GUELPA,B., The genoine of a baculovirus infecting Tipula paludosa (Meig.) (diptera): a high molecular weight closed circular DNA of zero superhelix density. Virologg, 1979, 96, 633-639. SCHNEIDER,I., Differentiation of larval Drosophila eye-antennal discs in vitro. J. exp. Zool., 1964, 156, 91-104. SCHNEIDER, I., Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embrgol. exp. Morphol., 1972, 27, 353-365. SHELDRICK, P., LAITHIER, M., LANDO, D. & I~YHINER, M.-L., Infectious DNA from herpes simplex virus: infectivity of double and single stranded molecules. Proc. nat. Acad. Sci. (Wash.), 1973, 70~ 3621-3625. SHURE, M. & VINOGRAD, J., The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method. Cell, 1976, 8, 215-226. SINGH, K. R. P., Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aeggpti (L.), Curt. Sci., 1967, 36, 506-508. STow, N. D. & WILKIE, N. M., An improved technique for obtaining enhanced infectivity with herpes simplex type 1 DNA. J. gen. Virol., 1976, 33, 447-458. SUMMERS,M. D. & ANDERSON, D. L., Characterisation of nuclear polyhedrosis virus DNAs. J. Virol., 1973, 12, 1336-1346. TWEETEN, K. A, BULLA, L. A. & CONSmLI, R. A., Characterisation of an extremely basic protein derived from grauulosis virus nucleocapsids. J. Virol., 1980, 33, 866-876. VAUGHN, J. L., GOODWIN, R. H., THOMPI~INS, G. J. & McCAwLEY, P., The establishment of two cell lines from the insect Spodoptera /rugiperda (Lepidoptera: Noctuidae). In vilro, 1977, 13, 213-217. WANG, J. C., Variation of the average rotation angle of the DNA helix and the superhelical turns of covalently closed cyclic k DNA. J. tool. Biol., 1969, 89, 783-801. WOLF, K. & QUIMBY,~{. C., Established eurythermic line of fish cells in vitro. Science, 1962, 135, 1065-1066. YASUMURA, T. & KAWAKITO, Y. (no title). Nippon Rimsho, 1963, 21, 2101. ZWILLENBURG, L. 0. & WOLF, K., Ultrastructure of lymphocystis virus. J. Virol., 1968, 2, 393-399.