JUlY 1992, p. 4339-4347 0022-538X/92/074339-09$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 66, No. 7

JOURNAL OF VIROLOGY,

Generation and Analysis of Defective Genomes of Autographa californica Nuclear Polyhedrosis Virus HO YUN LEE AND PETER J. KRELL* Department of Microbiology, University of Guelph, Guelph, Ontario, Canada NIG 2WI Received 26 November 1991/Accepted 17 April 1992

We have generated defective genomes of Autographa californica nuclear polyhedrosis virus (AcNPV) by serial, undiluted passage in IPLB-SF-21 cell culture in an attempt to identify potential cis-acting sequences important for AcNPV DNA replication. Viral DNA isolated from some of the 81 serial passages was analyzed by pulsed-field gel electrophoresis, restriction endonuclease analysis, and Southern blot hybridization. AcNPV-defective genomes appeared to be generated through a series of successively smaller and transiently stable intermediates. Although the defective genomes at passages later than passage 65 (P65) were somewhat heterogeneous in size, those of the majority of the population had a mean size estimated to be 50 kb, or 40%o of that of standard virus. Defective genomic DNA at P81 hybridized strongly only to a 2.8-kb region mapping within 85.0 to 87.2 map units of AcNPV DNA (most of HindIII-K and a small part of HindIII-B), suggesting that the majority of P81-defective genomes were missing most of the 128-kb wild-type DNA sequence, except for this small 2.8-kb fragment. Furthermore, our results indicated that the defective genomes of P81 were composed largely of reiterations of this sequence. We suggest that the 2.8-kb DNA segment retained by the defective AcNPV genomes of P81 contains an important cis-acting element(s) sufficient for viral DNA replication in AcNPV-infected cells. The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has a 128-kb, double-stranded DNA (9, 29, 36) genome which is often altered by serial propagation of the virus in cell culture (6, 27, 34) or by mutagenesis (5). Genomes of such virus mutants, identified by phenotypic differences observed in the polyhedral inclusion body (polyhedra), often contained moderately amplified DNA sequences without major deletions. However, major deletions of the AcNPV genome were reported by Carstens (7) and Kool et al. (25). The genome of one polyhedral morphology mutant, M5, has a continuous deletion from 2.6 to 46.0 map units (m.u.) on the AcNPV physical map (41) and exists in a circular form with only 58% of the standard genome size, as determined by electron microscopy (7). Using continuous infection of SF-21 cells with a recombinant AcNPV in a bioreactor, Kool et al. (25) demonstrated the generation of defective interfering (DI) particles, in which the genome had a deletion from 1.7 to 45.0 m.u. For many animal viruses, the defective genomes (DGs) of DI particles from relatively high passage numbers ultimately retain only the cis-acting elements that are essential for genome replication and possibly packaging signals, while all or most of the trans-acting factors are supplied by the standard helper virus (3, 18, 21, 23). Moreover, although a large portion of the viral DNA sequences might be deleted, the cis-acting sequences retained are often heavily reiterated and the size of the resultant DG is often close to that of the standard virus genome (21, 24, 42). The existence and location of the cis-acting replication origins in the herpes simplex virus (HSV) genome were first inferred from studies of HSV DGs and DI particles (14, 18, 42). We wanted to identify cis-acting sequences essential or sufficient for AcNPV DNA replication by first generating DGs and identifying the conserved sequences in the DGs. Under our experimental conditions, several major alter*

ations of the viral genome were observed during 81 consecutive undiluted passages. The first alteration, identified at passage 15 (P15), persisted at least until P40 and consisted mainly of deletions of certain parts of the genome. The second fundamental change was initially observed at P35 to P40, and there was a continuous evolution of this change to around P55 to P65, at which time it became relatively stable, at least up to P81. This second alteration involved deletion of most of the genome and an amplification of a remaining small DNA segment. Although the population of DGs consisted of heterologous DNA segments, the DGs of P81 retained one predominant segment which originated from the HindIII-K region of the standard AcNPV genome. Moreover, this particular DNA segment was very small (approximately 2.8 kb) and appeared to be repeated several times to generate DGs about half the size of the original standard genome. MATERIALS AND METHODS

Cells, viruses, and insects. Spodoptera frugiperda IPLBSF21 (SF-21) cells (40) were maintained in Grace's medium (17) supplemented with 0.25% (wt/vol) tryptose broth and 10% (vol/vol) heat-inactivated fetal bovine serum. In some cases, amphotericin B (2.5 ,ug/ml), streptomycin (50 ,ug/ml), and penicillin (50 U/ml) or gentamicin sulfate (50 ,ug/ml) were added. The wild-type AcNPV E2 strain (36), which we used as the standard virus, was plaque purified (4) three times. Larvae of Heliothis virescens (tobacco budworm) were maintained on an artificial diet as previously described (26). Isolation of viral DNA. For large-scale purification of AcNPV, polyhedra were isolated from sixth-instar H. virescens larvae infected with AcNPV. Isolation of polyhedra and purification of virus and viral DNA were essentially as described by Arif and Brown (2). DNA from extracellular virus (ECV) from infected SF-21 cells was isolated as described by Summers and Smith (37). The method for the isolation of DNA referred to herein as intracellular virus (ICV) DNA was adapted from those described by Hirt (22)

Corresponding author. 4339

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and Summers and Smith (37). Briefly, the cell monolayer was treated with cold 0.1 to 1.x lysis buffer (1.Ox lysis buffer is 30 mM Tris [pH 7.5]-10 mM magnesium acetate1.0% Nonidet P-40), and the detached and lysed cells were transferred to a tube and kept on ice for 5 min with intermittent vortexing (four to five times) for about 15 s each time. Nuclei were collected by centrifugation for 5 min at 500 x g at 4°C. The nuclear pellet was washed three times in cold calcium- and magnesium-free 1 x phosphate-buffered saline (PBS) and resuspended in a minimal volume of PBS containing 40 mM EDTA, pH 8.0. A 5x Hirt solution (1x Hirt solution is 50 mM Tris [pH 8.0]-20 mM EDTA-1.0% sodium dodecyl sulfate-200 pLg of proteinase K per ml) was added slowly, to a final concentration of 1 x. After a 10-min incubation at room temperature, 5.0 M NaCl was added, to a final concentration of 1.0 M, and the lysate was kept on ice for 1 h or longer. The DNA-containing supernatant was collected after centrifugation for 30 min at 104 x g at 4°C and extracted with phenol-chloroform-ether. DNA was ethanol precipitated or subjected to equilibrium ultracentrifugation in cesium chloride-ethidium bromide in a Beckman vertical rotor as described by Sambrook et al. (35). Generation of DGs. SF-21 cells were subcultured to about 75% confluency and infected initially at a multiplicity of infection of 103 PFU per cell. After 3 days, 2 ml of undiluted supernatant from the first infection was used for the next passage to infect 8 x 106 cells in an 80-cm2 tissue culture flask in a final volume of 12 ml per flask. This procedure was carried out every 3 days for up to 81 consecutive passages. For analysis of the DG at a particular passage, 1 ml of the extracellular medium was used to infect 1.6 x 107 cells in a 175-cm2 flask. ECV and ICV DNAs were extracted at 2 to 3 days postinfection (p.i.) as described above. Pulsed-field gel electrophoresis. Agarose plugs containing DNA were prepared essentially as described by DeLange (10), with some modifications. Briefly, after they were equilibrated to 43°C, cells or nuclei were mixed with low-meltingtemperature agarose also at 43°C to a final concentration of 0.6% agarose. The nuclei, embedded in the solidified agarose plug, were then lysed at room temperature for 24 h in either 1 x Hirt solution containing 200 ,ug of proteinase K per ml or plug lysis buffer (1 x plug lysis buffer is 1.0% sarcosyl-200 ,ug of proteinase K per ml-0.18 M EDTA-10 mM Tris [pH 7.5]), which was changed twice during this time. The plug, equilibrated in electrophoresis running buffer, was inserted into the well of a precast gel containing 1.0% Rapid agarose (GIBCO-Bethesda Research Laboratories [BRL]) in running buffer (0.5 x TBE) (1x TBE is 75 mM Tris-25 mM boric acid-0.1 mM EDTA [pH 8.9]). Pulsed-field gel electrophoresis was in a contour-clamped homogeneous electric field apparatus (GIBCO-BRL) with buffer circulation at 4°C, at a constant 160 V and with variable pulse times of 90 s for the first 2 h followed by 15 s for the final 20 h. A A DNA polymer marker (Promega) was used as a DNA size reference. Preparation of DNA probes and Southern blot analysis. For preparation of the P40 and P81 DG DNA probes, the respective viral DNAs were first subjected to pulsed-field gel electrophoresis (Fig. 1). DNA bands migrating between 70 and 100 kb (for P40) and between 30 and 100 kb (for P81) were isolated from the resultant pulsed-field gels. DNA was then purified from the isolated gel fragments with a Geneclean kit (Bio 101, Inc., La Jolla, Calif.). All DNA preparations were labelled to high specific activities with [3 P]dCTP or [32P]dATP (3,000 Ci/mmol; Amersham Corp.) by a random primer extension method (13) according to the instruction of the manufacturer (GIBCO-BRL). Purified DNA was

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FIG. 1. Pulsed-field gel electrophoresis of AcNPV standard DNA and DGs at different passage numbers. Agarose plugs containing nuclei for ICV DNA were prepared and DNA solubilized as described in Materials and Methods. After pulsed-field gel electrophoresis, Southern blot hybridization was with [32P]dCTP-labelled standard AcNPV DNA. The sizes indicated in lane S are in kilobases, relative to lambda DNA polymers. Lane SF is DNA from uninfected SF-21 cells, and lanes P1 to P81 indicate the passage numbers. ori, the position of the wells; arrowheads, relative positions of bands in lanes P15, P40, and P65 analogous to those labelled to the right of lane P81.

digested with the appropriate restriction endonucleases under conditions specified by the manufacturer (GIBCO-BRL) and fractionated by agarose gel electrophoresis. Unless otherwise indicated, electrophoresis was at 2 V/cm for 16 h in 0.6% agarose gels at room temperature in 1 x Tris-acetateEDTA (TAE) buffer (1x TAE is 40 mM Tris-acetate-1 mM EDTA [pH 8.0]). Capillary transfer of DNA to Nytran membranes and subsequent hybridization at 42°C in 50% formamide was as described by Anderson and Young (1). For reuse of the membranes, the probe DNA was stripped from the Nytran membrane under alkaline conditions (30). RESULTS Changes in polyhedral phenotype with serial, undiluted passage. For passages up to P20, approximately 80% of the infected cells producing polyhedra had a phenotype of many polyhedra (30 or more polyhedra per cell). With continued passage, the percentage of cells with 30 or more polyhedra per cell declined while that of cells with the phenotype of few polyhedra (10 or fewer polyhedra per cell) increased up to a maximum proportion, at around P40, of approximately 80% among the cells producing polyhedra. After P40, the number of polyhedra per cell declined and some of the infected cells did not produce any polyhedra. By P50, greater than 90% of the cells producing polyhedra contained only one or two polyhedra per cell, and by P60, almost none of the infected cells produced any polyhedra by 3 days p.i., although most of them showed cytopathic effects by 5 to 7 days p.i. Genome size at different passages. Pulsed-field gel electrophoresis of P1 AcNPV DNA resulted in a major band migrating at approximately 130 kb (Fig. 1, lane P1), which presumably represented the full-length, 128-kb AcNPV DNA. By P15, in addition to this band, three smaller DNA molecules (arrowheads in Fig. 1, lane P15; individual, more discrete bands were visible in an autoradiogram exposed for

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FIG. 2. Changes in the HindlIl digestion pattern of AcNPV DNA upon serial undiluted passage. (A) Agarose gel of ICV DNA isolated at fifth passage digested with HindIII and stained with ethidium bromide. The Southern blot of gel A was probed with either P40 (B) or P81 (C) DNA isolated from pulsed-field gels and labelled with 32p. Lane M in panel A is the 1-kb DNA marker (GIBCO-BRL), with sizes of DNAs in kilobases indicated in lanes S. Letters to the right of lanes P0 indicate the position of the bands for the corresponding Hindll restriction enzyme fragments of AcNPV DNA. Arrowheads point to novel bands which contributed to the initial stages of the major changes of the band pattern. Less DNA was loaded for P60 in order to better resolve the band pattern for later-passage DNAs which generally had a higher hybridization intensity with later-passage DGs. Different exposures of the autoradiograms showed that the band patterns from P55 to P65 were essentially the same (data not shown). The numbers with arrowheads to the right of lane P0 in panels B and C refer to the positions and sizes (in kilobases) of novel DNA bands detected by hybridization with P40 or P81 DNA. every

a shorter length of time) migrating at approximately 100, 85, and 70 kb were observed. Hybridization to these four bands was also observed by P40, although the majority of the hybridization shifted from the 128-kb band to one in the 85-kb region. The passages at which hybridization to these four bands first appeared and subsequently declined coincided with those at which the proportion of cells having 10 or fewer polyhedra per cell of infected cells first started to increase and later to decrease, along with the appearance of no polyhedra in infected cells. The band pattern continued to evolve with further passages, and by P65 much of the hybridization seemed to shift from the four bands to a new, dominant, faster-migrating, broad band of viral DNA (Fig. 1, lane P65). The majority of viral DNA molecules from P65 to P81 ran as a broad band centering around the 50-kb position but with an overall range from 30 to 100 kb. The hybridization band pattern of ICV DNA from P81 was essentially the same as that for P65 (Fig. 1, lanes P65 and P81). In a separate experiment, the ECV at P81 contained, in addition to the 128-kb DNA species, a 50-kb DNA species. This smaller DNA may be from DI particles, but the relative amount of this smaller DNA in the ECV fraction was much lower than that for the P81 ICV DNA (data not shown). The passage at which the broad 50-kb DG DNA band first appeared coincided with the earliest passage at which none of the infected cells produced polyhedra by 3 days p.i. Deletion and amplification of AcNPV DNA fragments with continued passage. In order to investigate the specificity of the genome alteration during serial passage, the restriction enzyme patterns of the ICV DNAs were analyzed at every fifth passage (Fig. 2). The overall restriction enzyme patterns

remained unchanged up to P10, but differences were detected by P15 and were more clearly detectable by P20. One such change was the appearance of a new 14.5-kb HindIII fragment which was easily observable by P20 (Fig. 2A) but which was first detected by P15 on an overexposured autoradiogram (data not shown). The appearance of this new band coincided with a noticeable reduction in the relative intensity of the HindIII-A/B band (Fig. 2A). Similarly, a new, larger PstI DNA fragment (approximately 30 kb) was first observed around P25 (data not shown). By P30, there was a decrease in the relative hybridization to PstI and EcoRI fragments (PstI-B, -E, -F, and -G and EcoRI-A, -D, -J, -K, -L/M, -N/0, and -UIV) mapping between 8.0 and 37.7 m.u. and between 62.3 and 97.0 m.u. (data not shown). A second significant alteration was first observed at around P35 to P40, when novel HindIII fragments at 1.74 and 1.80 kb appeared. The relative intensities of these bands increased with subsequent passages (Fig. 2A). Because the molar ratios of the 1.74- and 1.80-kb HindIII fragments were much higher than those of any others, the viral DNA sequences represented by these fragments were probably highly amplified after P40. A reduction in the relative amount of DNA in bands such as HindIII-A/B, -D/E, -I, -J, -L, -M, -N, and -O was observed around P40, and a similar reduction for HindIII-F, -R, -S, -T/U, -V, and -W/X was noted around P50 (Fig. 2A). The relative proportions of these and most of the other fragments continued to decrease with later passages. On the other hand, the relative intensity of the HindIII-K band increased slightly at later passages (Fig. 2A). The passages at which specific alterations in restriction enzyme patterns first appeared (Fig. 2A) at both early

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(around P15 to P30) and late (P40 to P65) passages correlated to passages at which changes in the gel profiles of undigested DNA were also first observed by pulsed-field gel electrophoresis (Fig. 1). The organization of P40 and P81 DGs is different. In order to determine which part of the standard viral DNA genome remained in the DG DNA from P40 and P81, a Southern blot of HindIII-digested ICV DNA isolated from infected cells at different passage numbers (Fig. 2A) was subjected to hybridization with either 32P-labelled P40 DNA (Fig. 2B) or 32p_ labelled P81 DNA (Fig. 2C) probes, both of which were isolated from pulsed-field gels. P40 DNA hybridized strongly to HindIII-A/B, -C, and -K, at about equivalent relative intensities. Hybridization was also found with bands at 1.74, 1.80, 2.0, 2.3, 3.2, 3.5, and 14.5 kb. Weak hybridization signals were observed with HindIII-F, -G, and -H. Although the hybridization pattern with 32P-labelled P81 DNA (Fig. 2C) was somewhat similar to that with P40 DNA (Fig. 2B), three specific differences were noted. First, hybridization to the HindIII-C band which was seen with P40 DNA was not detected with the P81 DNA probe. Second, the degree of hybridization to HindIII-F with P81 DNA was stronger than that with P40 DNA. Third, P81 DNA hybridized more strongly to HindIII-K (2.9 kb) than to HindIII-A/B, -C and the 14.5-kb bands, and by P35, it hybridized to smaller DNA fragments of 1.25, 1.74, 1.80, 2.0, 2.3, 3.2, and 3.5 kb. Weaker hybridization between the P81 probe and latepassage DNA (P45 to P65) was also observed for a series of fragments larger than 4.7 kb which formed part of a ladder of bands starting from the 1.74-kb band and increasing up to a viral DNA band at 14.5 kb (Fig. 2C; lane P60 contained less DNA to better resolve these hybridization bands). The hybridization patterns for P40 and P81 DNAs suggested that P40 DNA contained DNA from HindIII-C and -K and the 14.5-kb DNA in almost equimolar amounts, and since no hybridization was detected between P81 DNA and HindIII-C, most of the DGs of P81 did not contain this fragment, while at least a portion of the HindIII-B sequence was retained. Since P40 DNA hybridized strongly to HindIII-C but the P81 probe did not, this DNA sequence must have been lost after P40. The most intriguing observation was that most of the P81 DNA retained an unaltered HindIII-K fragment, while most other parts of the genome were apparently deleted or otherwise changed. DGs are relatively stable after P65. The overall restriction endonuclease profile did not seem to change very much between P55 and P65 (Fig. 2). To determine whether further alterations could occur after P65, we compared P65 and P81 DNAs (Fig. 3A and B). For P65 DNAs, the HindIII digestion patterns and the Southern blot hybridization results that used standard AcNPV and P81 DNA as probes appeared to be very similar to those for P81 DNA, suggesting that the DG after P65 was relatively stable, at least up to P81. This observation is consistent with the similarity in the gel profiles of uncleaved P65 and P81 DNAs after pulsed-field gel electrophoresis (Fig. 1). Hence, the P81 DG might represent the ultimate minimal DG which had evolved by P65 from standard AcNPV under our conditions of serial, undiluted passage. DGs likely consist of a heterologous population of viral DNA. Figure 3B also showed that the XhoI and HindIII digests of P81 DNA generated numerous fragments, from 0.55 to 5.9 kb, which formed a ladder of bands in which the relative intensities of hybridization with P81 DNA to each band were variable. The most intense hybridization was with

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FIG. 3. Comparison of the DNAs from PO, P65, and P81. Southern blot hybridization of AcNPV ICV DNA isolated at PO (standard AcNPV), P65, and P81 and digested as indicated above each lane with HindIll (+H), EcoRI (+E), and XhoI (+X) was with either 32P-labelled standard AcNPV DNA (A) or P81 DG DNA (B). In lanes H, E, and X, the letter designations are for the restriction endonuclease fragments for HindIll, EcoRI, and XhoI, respectively, as indicated above each lane. The numbers to the left of lanes P65+H and to the right of lanes PO+X indicate the positions of novel bands in lanes P65 and P81.

P81 XhoI DNA fragments with sizes of 1.2, 1.5, 1.74, 1.80, 3.0, and 3.6 kb. For both digests, the larger DNA fragments did not hybridize as strongly as the shorter fragments with labelled P81 DNA. It therefore appeared that the P81 DG consisted of a diverse population of viral DNA genomes heterologous with respect to size and organization. This interpretation is also supported by the analysis of uncleaved DG DNAs after pulsed-field gel electrophoresis showing a spread of viral genomic DNAs into a rather broad band (Fig. 1). A majority of the P81 genomes also seemed to have retained only a small proportion of the original standard DNA segments, since P81 DNA hybridized to only certain discrete bands from digested standard AcNPV DNA (Fig. 3B, P0 lanes; Fig. 4). DGs of P40 contain two separate regions of the standard AcNPV genome. To identify the DNA sequences present in P40 and P81 DNAs, we compared hybridization between restriction endonuclease-digested standard AcNPV DNA and 32P-labelled P40 and P81 DNAs (Fig. 4). From Fig. 4B and the summary of the hybridization results in Fig. 5 and 6, it appeared that major portions of the standard DNA were missing from most of the P40 DGs. In particular, KpnI-C, SacI-C, -E and -F, PstI-E to -0, and BglII-F fragments did not hybridize at all or hybridized only weakly with P40 DNA. These observations suggested that this apparently deleted region extended from at least 3.62 to 43.7 m.u. (40% of the standard AcNPV genome in size). In addition to the fragments noted above, several other fragments which mapped entirely within this region also hybridized only weakly or not at all with the P40 probe. A region which was represented by DNA fragments KpnI-D and -E, BstEII-G,

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EcoRI-E and -L, XhoI-G, and BamHI-E and -G and which spanned the genome from 65.2 to 82.3 m.u. also appeared to be deleted. The combined regions deleted from the standard viral DNA sequence in the P40 genomes therefore represented at least 57.4% of the standard genome. If the sizes of these deletions are compared with those of the 100-, 85-, and 70-kb DG DNA species observed from the pulsed-field gel electrophoresis experiment (Fig. 1), the simplest interpretation would be as follows. The 85-kb DNA species, which is the most abundant by P40 (Fig. 1, lane P40) and is approximately 65% of the full-length standard virus genome in size, might represent AcNPV DGs having a deletion from 3.62 to 43.7 m.u. (leaving approximately 60% of the standard virus genome). Similarly, the 100- and 70-kb DNA species (approximately 80 and 55% of the standard virus genome in size, respectively) might be equivalent to DGs with a deletion from 65.2 to 83.9 m.u. (19% deletion) and those with a deletion of both regions (a total deletion of 59%), respectively. The same hybridization experiments also suggested that for P40 there were two distinct groups of DNA fragments having different relative hybridization intensities. A high level of hybridization was observed (Fig. 4B) with DNA fragments SacI-D and SmaI-C (46.7 to 64.8 m.u.) and XhoI-I (81.6 to 86.4 m.u.), and some was observed with XhoI-J (86.4 to 89.0 m.u.). These two regions together constituted approximately 25.5% (32.6 kb) of the total standard AcNPV genome. HindIII-F and -G (90.4 through 100 and 0.0 to 3.3 m.u.) also hybridized to the P40 DNA probe. A group of DNA fragments flanking this region, including HindIII-P and -Q, XhoI-K, and BamHI-D and -F, also showed weak hybridization with the P40 probe, suggesting that P40 DGs retained, as a minor component, standard AcNPV DNA sequences spanning from 82.3 through 100.0 and 0.0 to 4.8 m.u. The identification of at least three major groups among the P40 DGs, each with a different hybridization pattern of DNA segments, supports the idea that the P40 DGs consist of a heterologous population of viral DNAs of different sizes (Fig. 1, lanes P65 and P81).

A majority of P81 genomes contained only a very minor proportion of the standard AcNPV DNA. The results of Southern blot hybridization between P81 and standard virus DNAs (Fig. 4C and Fig. 6) showed that the P81 DNA did not hybridize to restriction endonuclease fragments HindIII-V, KpnI-C, SacI-C and -F, and SmaI-D, suggesting that these were absent from the DG by P81. Similarly, all other fragments from within this region and mapping between 3.3 and 49.4 m.u. did not hybridize with the P81 DNA probe, suggesting that most of this continuous region was deleted by P81. Deletions involving fragments HindIII-C, XhoI-B and -G, and BamHI-G were also apparent by the lack of hybridization between P81 DNA and these fragments from standard AcNPV DNA, suggesting a continuous deletion between 53.3 and 82.3 m.u. The strong hybridization signal observed with the P81 DNA probe was almost exclusively with HindIII-K (85.1 to 87.2 m.u.) and fragments which overlap HindIII-K, including PstI-B, BamHI-B, SacI-A and -B, SmaI-A, KpnI-B, BglII-E, BstEII-C, EcoRI-H, and XhoI-I. Although EcoRI-F, G, and H comigrate and XhoI-H and -I comigrate, the mapping with other enzymes suggested that the major hybridization of the P81 DNA probe occurred only with EcoRI-H and XhoI-I from these triple- and doublefragment bands, respectively. HindIII-A/B also showed fairly strong hybridization with P81 DNA, most of which could have been to HindIII-B (68.2 to 85.1 m.u.), which is adjacent to the HindIII-K region that hybridized very strongly to the P81 probe. Probably, some additional hybridization was also due to HindIII-A (37.7 to 53.3 m.u.), which hybridized weakly with the P81 probe (see below). However, only weak signals were seen with BamHI-D (82.3 to 85.0 m.u.) to the left and HindIII-Q (87.2 to 88.8 m.u.) to the right of the HindIII-K fragment. Therefore, the majority of the DGs in the mixed population of P81 DG DNA retained not more than 2.2% (2.8 kb) of the total standard AcNPV genome spanning from 85.0 m.u. (right-hand limit of BamHI-D) to 87.2 m.u. (left-hand limit of HindIII-Q and right-hand limit of HindIII-K). However, the fact that weak

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FIG. 5. Summary of the mapping of the DGs of P40 and P81 DNAs to the standard AcNPV genome. The parental DNA sequences which were retained in the DGs of P40 and P81 were determined by Southern blot hybridization (Fig. 4), and these are indicated relative to the physical map of the standard AcNPV genome. Filled bars indicate DNA segments which map to fragments that hybridized strongly, while hatched bars indicate adjacent fragments which also hybridized, but at a lower intensity, to 32P-labelled P40 or P81 DNA, as indicated. + denotes other DNA fragments which hybridized, but only weakly, to the probes. Single lines bracketed by upward-facing arrows indicate the regions which appeared to be missing from the respective DGs, since the DG DNA did not hybridize to fragments within these regions. The regions identified in panel A as A to F are enlarged in panel B. A, C, and E are for P40 DNA while B, D, and F are for P81 DNA. Sc, Sacl; Sm, SmaI; E, EcoRI; B, BamHI; H, HindIII; X, XhoI; Bs, BstEII; R, EcoRV; Bg, BglII; P, PstI; K, KpnI; Hin A, HindIII-A; Hin C, HindIII-C; Hin G, HindIII-G; Hin F, HindIII-F. Not all of the restriction endonuclease sites are shown. Numbers below B, D, and F in panel B are in map units.

hybridization with P81 was also observed with fragments from either side of HindIII-K suggested that at least some of the P81 DGs also retained longer regions which could have included some DNA from as far left as BamHI-D (to 82.3 m.u.) to as far right as HindIII-Q (to 88.8 m.u.). Some of the labelled P81 DNA also hybridized, although only weakly, to fragments mapping to two widely separated regions of the standard virus genome (Fig. 4C and Fig. 6). Since P81 DNA hybridized to fragment SacI-D but not to SmaI-D or HindIII-C, the part of the SacI-D region which hybridized only weakly to P81 mapped to between 49.4 m.u. (right end of SmaI-D) and 53.3 m.u. (left end of HindIII-C). SmaI-C, which overlaps with SacI-D, also hybridized weakly with the same probe. Another region which was apparently retained as a minor component mapped to 97.0 through 100.0 and 0.0 to 3.3 m.u. For fragments within this region, HindIII-F and PstI-N (Fig. 4C) hybridized to the P81 DNA but HindIII-G did not. Other fragments which overlap with HindIII-F, such as BstEII-D, BglII-C, PstI-D, and XhoI-C, also hybridized weakly to the P81 DNA probe.

These two weakly hybridizing regions together retained not more than 10.2% (13.1 kb) of the parental DNA. It is unlikely that the weak hybridization to these regions was due to sequence homology between the major P81 DNA component (HindIII-K) and those two regions, because cloned HindIII-K did not hybridize to these regions (data not shown). P81 DGs did not contain chromosomal DNA. Southern blot hybridization with AcNPV DNA as a probe showed that, even after prolonged exposure, no hybridization was observed between AcNPV DNA and SF-21 chromosomal DNA isolated from uninfected cells (Fig. 1, lane SF). Similarly, no hybridization was observed between 32P-labelled gel-purified P81 DG DNA and SF-21 chromosomal DNA (Fig. 2C, lane SF and data not shown). In accordance with these observations, 32P-labelled chromosomal DNA did not hybridize to intracellular P81 DG DNA which was isolated by cesium chloride-ethidium bromide equilibrium centrifugation (data not shown). These results therefore suggested that P81 did not contain detectable amounts of SF-21 chromosomal DNA. AcNPV homologous repeat sequences (hrs) were not amplified in P40 and P81. Analysis by Southern blot hybridization suggested that hrs were not amplified in later-passage DGs because 32P-labelled P40 DNA did not hybridize to either HindIII-L or BstEII-G, which contain hr2 and hr4, respectively, and only weakly hybridized to HindIII-F and -Q, which contain hrl and hr5, respectively (Fig. 4B). Only SacI-D, which encompasses hr3, hybridized strongly to the P40 DNA. However, since no other restriction endonuclease fragments known to contain an hr (such as HindIII-F, -L, and -Q and BstEII-G) hybridized strongly to P40 DNA, the strong hybridization signal to SacI-D probably was not due to any amplification of hr3 itself but rather was due to other sequences within this fragment. Similarly, P81 DNA did not hybridize to segments containing hr2 and hr4 (within HindIII-L and BstEII-G, respectively) and only very weakly hybridized to fragments bearing the other 3 hrs (HindIII-F for hrl, SacI-D for hr3, and HindIII-Q for hr5) (Fig. 4C). In contrast to the hybridization with the P40 probe, fragments containing the hr3 region hybridized only weakly with P81 DNA. Since hrs-bearing fragments hybridized only poorly, if at all, with P81 DNA, it is unlikely that hr DNA sequences were amplified in the DGs, and they might even have been deleted by P81.

DISCUSSION In order to have some insight into DNA replication of AcNPV, we wanted to first generate a smaller genome containing only minimal and essential cis-acting DNA segments required for DNA replication. We accomplished this by generating DGs through serial undiluted passage. Although we did not systematically test the interfering character of our late-passage particles, the generation of DI particles by continuous virus replication in a bioreactor (25) or serial passage of recombinant AcNPV (43) has been reported for AcNPV. Also, in a preliminary experiment that used late-passage virus, we noted a delay in the replication cycle and reduced severity of cytopathic effects in infected SF-21 cells, suggestive of DI particles. The three discrete bands, representing DNAs migrating at the equivalent of 100, 85, and 70 kb, were the first DGs observed after serial passage. Because these three bands persisted for more than 25 passages (at least from P15 to P40), they may have had some selective advantage over

DEFECTIVE GENOMES OF AcNPV

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FIG. 6. Hybridization summary for P40 and P81 DNA probes against standard AcNPV DNA. 40 and 81 above each column represent ICV DNA isolated from P40 or P81, respectively, and used as probes for hybridization. +, + +, and - denote weak, strong, and no hybridization, respectively, between the corresponding restriction enzyme (indicated above each column) fragment (Fgt) and probe. Comigrating bands are indicated by boxes, and fragments within a box are collectively marked +, + +, or -, which may not necessarily represent the relative degree of hybridization for each fragment within that particular box. +/- markers indicate hybridization signals which were above background levels but very weak.

standard virus DNA and were not simply a result of random alterations of the genome. With continued passages, a major shift in the size of the majority of the ICV DNA from a standard virus genome size (128 kb) to an estimated size of approximately 85 kb was observed. Hence, these smaller viral genomes may be the most stable intermediates up to P40 and may be equivalent to the DI genomes detected by others (7, 25, 43), while the other two species (70 and 100 kb) have not been reported previously. Since the major component of the latest passage, P81, is the HindIII-K sequence and since P81 DNA hybridized to all three bands in addition to the 128-kb standard virus DNA (data not shown), all three DG DNA species must have shared the HindIII-K sequence. Considering all of these data, the generation of a minimalsize DG seemed to be a gradual process, involving several successive deletions. One of the deleted regions that we noted for the P40 DGs (3.4 to 43.7 m.u.) overlaps with the largest deletions reported for the AcNPV genome as described by Kool et al. (25) (1.7 to 45.0 m.u.), Carstens (7, 8) (2.6 to 46.0 m.u.), and Wickham et al. (43) (from approximately 0 to 35 m.u.). Since a deletion of this entire region was observed from four independent studies, it is unlikely that any cis-acting sequences essential for DNA replication reside in this region. This deleted region included the genes coding for viral DNA polymerase (39.5 to 42.5 m.u.) (39) and viral proliferating cell nuclear antigen (within 29.0 to 30.5 m.u.) (33), both of which are presumably necessary for optimal DNA replication, albeit in trans. Sufficient virus DNA polymerase and proliferating cell nuclear antigen were probably supplied by standard helper AcNPV for this DG to replicate. The DNA

polymerase gene of HSV was similarly deleted in HSVdefective genomes (18). All or most fragments containing each of four (and possibly all five) hrs were deleted by P40 and were not detected by Southern blot hybridization with P81 DNA. Although the hrs act as enhancers in the AcNPV genome (19, 20) and transcriptional enhancers are often necessary for optimal initiation of DNA replication (11), for AcNPV the hr enhancers might not have a direct role in DNA replication. Even though more than 97% of the parental standard genome appeared to have been deleted in most of the P81 genomes, at least some of the DGs must have been encapsidated and released from the cells to contribute to the next infectious cycle. Although we detected a 50-kb DNA in the ECV fraction of P81, presumably as DIs (data not shown), it was a very minor component compared with that in the ICV fraction. Thus, either not all of the replicated DGs were encapsidated or all were encapsidated but most were not released as ECVs. Therefore, the DGs of later passages might have more of a replicative than a packaging advantage. The fact that HindIII-C, which includes the coding region for the major capsid protein (38), hybridized strongly to P40 DG suggested that capsid proteins might give the DG species a replicative advantage similar to that for simian virus 40 DIs containing the coding sequence for simian virus 40 major capsid protein (32). Perhaps the HindIII-C region, like the HindIII-K segment described below, contains a cis-acting element important for DNA replication and was therefore retained until at least P40. However, it might not have been as essential as the HindIII-K region and was therefore lost at later passages.

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The existence of some minor viral DNA classes in the P81 DGs was suggested by the observation that in addition to some regions, such as HindIII-K, which hybridized strongly, others, such as SmaI-C, also hybridized (although weakly) to labelled P81 DNA. The standard viral DNA sequences retained as a minor component in a proportion of genomes in P81 presumably also have some selective advantage, perhaps by providing some trans-acting factors or cis-acting sequences such as those required for packaging or acting as termini for DNA replication. Although the P81 DG DNA was heterologous in size, the median size was 50 kb. This is about 17 times larger than the 2.8-kb DNA segment which appeared to be the only major parental viral DNA sequence retained by all or most of the P81 DG genomes. Since DG DNA did not appear to contain chromosomal DNA, it is likely that the P81 DG consisted of many reiterations of the 2.8-kb DNA segment (or a part of that segment), with the number of repeats dictating the ultimate size of the DGs. There may be a minimal physical DNA size needed to form a stable nucleocapsid and hence a DI particle. The minimal 2.8-kb DNAs efficient for DNA replication would then have to be repeated until a large enough defective DNA is formed for encapsidation. As shown with DI particles in other viruses, repeated sequences might confer a replicative advantage (28). Because the parental AcNPV genome had lost most of its DNA, except for the 2.8-kb fragment, in the process of generating a DG, presumably this small DNA segment retained only the cis-acting sequence(s) sufficient and essential for DNA replication. Cloning and sequence analysis of some of the abundant restriction endonuclease fragments of the later DGs are currently in progress in our

laboratory. Among the parental standard AcNPV DNA segments retained by the P81 genomes, the major portion (2.8 kb) mapped to the well-characterized (15, 16, 31) HindIII-K region (Fig. 4C and 5). The left end extended into HindIII-B and included the 3' cotermination sites for the nine mRNAs

reported by Oellig et al. (31). All of these overlapping mRNAs are transcribed from left to right on the physical map and end near 85.0 m.u. The HindIII-K region itself is believed to encode 35- and 94-kDa proteins, and transcription of these immediate-early genes starts at around 87.0 m.u. but proceeds in opposite directions (15, 16). The gene for the 35-kDa protein is crucial for normal viral replication in cultured cells (12), while the 94-kDa protein might not be required for virus replication (16). In addition to these two gene transcripts, the authors identified several additional transcripts transcribed in both directions, including one which was transcribed very strongly late in infection. Since these later transcripts did not appear to code for any proteins, Friesen and Miller (16) speculated that they might regulate transcription. We believe that the high level of transcriptional activity and utilization of different promoters at different times after infection, which is a feature of this region, might also influence DNA replication, perhaps at the level of initiation, as has been shown for other viruses (11). The DNA from the HindIII-K region which was retained by P81 was also the major AcNPV DNA component in the ladder of bands revealed after hybridization of P81 DNA digested with either HindIII (Fig. 2C) or Xhol (Fig. 3B). The DNA band pattern in the ladder showed a certain regularity, indicating that the DGs have discrete rather than random sizes of insertions or numbers of repeats. A similar band pattern was observed with HSV type 1 Angelotti DI DNA, in which the regularity of the band pattern was due to insertions of increased numbers of a packaging, terminal DNA

sequence (24). This report may relate to our own observations, according to which the DNA bands higher in the ladder were less abundant than those in the smaller-size range. We suspect that in cells carrying the DGs, there are genomes with a variable number of inserts of a specific segment from the HindIII-K region of the genome. The existence of such a minor component of DNA sequences observed with the P81 genome might be responsible for forming the ladder-type bands which hybridized with the

HindIII-K or P81 DNA probe. ACKNOWLEDGMENTS We are grateful to Peter Dobos for critically reviewing the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada (grants OGP0008395 and STR 0101482) and the Canadian Forestry Service (PRUF grant 01K38-6-

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