Physical mapping of Malacosoma neustria nuclear polyhidrosis virus genome and its modification in Antheraea pernyi cell culture

A physical map of the M. neustria nuclear polyidrosis virus (ManeNPV) genome was constructed, the complete order of BamHJ, Kpnl and Pstl restriction enzyme sites was determinedt a polyhedrin gene was localized on the map. The viral DNA size was calculated to be about 139 kbp. Restriction endonuclease profiles of the DNA of ManeNPV plaques isolate propagated in A. pernyi cells demonstrated ^persistent heterogeneity*, submolar bands were shown to appear in a digestion pattern of DNA of the first passage virus. These bands were proved to be due to the DNA molecules presence in the non-homogeneous virus DNA pool, their chains having been shown to carry a putative break in a definite site. Such a *break site» was localized on the physical, map of ManeNPV genome.

For example, during such an outbreak in the middle of the XX century many thousands of hectares of Eastern Europe woods were completely deprived of leafage.Currently, Mane populations are controlled by the application of chemical insecticides.
In our laboratory a virus strain has been isolated from a diseased Mane larvae [4 ].M. neustria nuclear polyhedrosis virus (ManeNPV) is a species in the genus Nucleopolyhedravirus, family Baculoviridae.It has been adapted to the cell line MCAp-1 derived from A. pernyi larvae [5 J. Later the ManeNPV polyhedrin gene has been localized in an EcoRI-A fragment of virus genome [6 ] and complete sequence of this gene has been determined.High productivity of ManeNPV in cultivated MCAp-1 cells as well as a high level of polyhedrin expression in the late infec tion stage have made this cell-virus system a good candidate for use as an expression system for recom binant protein production under the polyhedrin gene promoter control [7,8 ].Working with ManeNPV we have detected its interesting feature.Submolar bands have been observed in the REN profiles of virus DNA indicating that the virus isolate used in this study is not homogenous and contains minor genotypic va riants.We have failed to select out a homogenous virus clone using routine multiple plaque-forming approach, minor submolar bands have been detected in the REN profiles of the DNA of all plaque isolates.
Aiming at usage of ManeNPV as an insecticide and a vector for recombinant protein expression as well as understanding the nature of its unusual «persistent heterogeneity* we have begun to study its genetic organization.
Materials and Methods.MCAp-1 cells were main tained in the Grace's medium supplemented with 10 % heat-inactivated fetal bovine serum.Some de tails of the cell cultivation, virus propagation, and plaque assay were described previously [9].Plaque assays were incubated for 4 days at 27 °С Infected cells and extracellular virus (ECV) fluids for viral DNA extraction, virus stock obtaining and virus titer estimation were made 7 days p. i.
Viral DNA from infected cells was isolated ac cording to the method previously described [10] with some modifications.This method permits to obtain supercoiled forms of viral DNA with minimal cellular DNA contamination.DNA was isolated directly from virus-infected cells without preliminary virus puri fication.To isolate each preparation, (5-8) • 10 6 cells were suspended in 100 pi of 0.05 M EDTA solution.An equal volume of 2 * buffer containing 50 mM EDTA, 0.5 mg/ml spermidine, 0.4 mg/ml ethidium bromide was then added.The pH of this mixture was adjusted to 11.5 with 0.25 M NaOH and then neutralized with 0.1 M HC1, 10 pi of RNAse A (10 mg/ml) was added.After incubation (37 °С, 30 min) sodium laurylsulphate was added to the lysate (1 %) as well as proteinase К (1 /ig/ml).The mixture was incubated overnight at 37 °С.DNA was extracted three times by phenol, one time by chlorophorm and then ethanol precipitated.Alternatively, viral DNA was isolated from infected cells and ECV according to the standard procedures described by O'Reily [9].
Viral DNA was digested with different restriction endonucleases and then run in 0.7 % agarose gel electrophoresis using Tris-borate buffer system [11].

Molecular sizes of the ManeNPV DNA fragments obtained after digestion have been estimated by comparison with phage DNA Hindi
IIIEcoRI/BamHI markers.For high molecular weight fragments the second enzyme was used to generate smaller frag ments and to provide better estimation.For this purpose viral REN fragments were separated in 0.7 % low melting agarose gel, gel slices including large fragments were excised, heated to 68 °С, two-fold diluted with ТЕ buffer and digested using appropriate buffer.
Results and Discussion.Our first step was the construction of the ManeNPV physical map and locating on this map the site the changes of which cause the appearance of submolar bands in electrophoregrams of the digested virus DNA.
One of two ManeNPV strains isolated from Mane larvae -the isolate K2 [4 ] -was selected for fur ther investigations.The REN digestion pattern of this isolate did not contain submolar bands, therefore we have supposed K2 to have a single genotype.How ever, the further REN digestion followed by the hybridization of blotted REN fragments with the 32 P-labelled DNA of the same virus demonstrated low intensity of submolar bands in the digested DNA patterns.As a result of more attentive study of the electrophoregrams obtained we have revealed such submolar bands also in the REN profiles formed after the excessive virus DNA introduction into sample wells.These data are against homogeneity of the clone obtained; so we purified it additionally, three plaque passages were made.Fifteen plaques were selected out during the third passage; each plaque variant was reproduced to obtain virus stocks.These stocks were used for the cell culture infection, the MOI being 5 pfu/cell.The viral DNAs obtained from the reproduced virus isolates were REN digested.We attempted to select out in such a way any virus isolate presenting either a single major or a single minor fraction of the K2 isolate.Among 15 REN digested virus DNA preparations 13 ones were shown to possess submolar fractions.Two virus isolates pre sented single major fraction following such a treat ment.However, after the second in vitro passage of the both isolates their DNAs were not homogenous.It should be noted that «homogeneity» or «heterogeneity» are not strict terms, so-called homogenous DNA samples may contain microquantities of the molecules of minor fraction undetectable by the following REN analysis.All 15 isolates demonstrated the identical heterogenous REN DNA profiles, the only difference concerned the submolar bands inten sity.An isolate, Wl (referred then as ManeNPV), was chosen among 15 ones for further investigations because of the higher intensity of its minor bands comparing to the minor bands of other isolates.Our investigations have shown the constant quantity and size of the ManeNPV submolar fragments for each restriction endonuclease used; they do not depend on  -A, В, С; KpnI-A, С, D arrow in fig. 3) which coincids with the PstI recog nition site.Co-location of the sites for three casually chosen restriction endonucleases has permitted us to suppose this point to be not a specific recognition site for each one, but a site of non-specific or specific virus DNA break.To confirm this idea, we had to locate in the virus genome some submolar fragments obtained after digestions with other restriction en donucleases.
The results of the blot-hybridization experiments (fig. 1, В, C) demonstrated without any doubt that the sums of minor fragments sizes obtained after the EcoRI and BgUI digestions corresponded to the sizes of higher molecular fragments hybridizing with 32 Plabelled BamHI minor fragments (10 kbp + 14 kbp e = 24 kbp for EcoRI; 2.0 kbp + 0.9 kbp -2.9 kbp for BgUI).Earlier we have mentioned the lack of the second submolar band in the Яшс/Ш-digested viral DNA electrophoregrams (fig. 1, Л We have found that the use of more «unfavorable» conditions of DNA purification (longer phenol-chloro form extraction procedure and more intensive mixing) causes the increasing of total quantity of damaged DNA molecules and, as a result, the smearing of the REN DNA profiles, the intensity of minor bands having, however, no tendency to increase.According to these data the most probable situation is the existence of a minor virus variant carrying breakcontaining genomes.Any two-stranded DNA break is known to disclose a supercoiled DNA and form linear DNA molecules.However, the circular DNA form is well known to be a natural feature of any baculovirus life cycle, no undamaged «naturab linear forms having been found for any baculovirus studied.Some facts suggest ManeNPV is a typical baculovirus with a typical genome.First, any virus DNA isolation using the alkali-ethidium approach permits to obtain only supercoiled molecules, all the linear ones being destroyed by the alkali treatment.The presence of the linear molecules in such samples is proved to be a result of the following chloroform-phenol deproteinization.Further, such break-containing viral genomes have been shown to be presented not only in the infected cells, which can contain any forms of viral DNA but in the ECV too.The linear viral genomes seem incapable to be packed into maturing particles and to form virions.These data and considerations develop a clear contradiction: it goes that the viral genome integrity is damaged but this genome exists simultaneously as a supercoiled structure.This con tradiction may be explained by a hypothesis that any DNA molecule of the minor genotypic virus variant really contains a break.However, the molecule is stabilized by a protein or proteins, hence a protein «Іоскег» ties both ends of the DNA molecules.Such protein (s) are destroyed during the DNA deproteinization and phenol-chloroform treatment causing the DNA linearization.
As we have already discussed usual accumulation of modified virus genomes in a virus clone seeming originally homogenous is a stepwise process following virus passages.The fact that the heterogeneity of the plaque ManeNPV isolates is seen in the first passage virus with low MOI testifies that the term «modification» is hardly applied to this phenomenon.The nature of changes detected here -a break at a determined point of the DNA sequence-permits to make an assumption, that it is not the modification in a strict sense of this word, which means some changes comparing with the natural state, but a phenomenon, which can reflect a natural step of the ManeNPV intracellular reproduction.Our data permit only to make some speculations and to suppose an important functional role of the break point -for example, during replication and/or recombination process or other processes requiring DNA strands break.Some reparation processes are possibly realized with low effectiveness in the virus-cell system studied here, so the break-containing DNA molecules are easily detec ted in the virus DNA pool.
Either the DNA molecule break is a result of high damage sensitivity of its locus during purification or it is masked by protective protein «naturally» present in baculovirus genome -in any case, this unusual phenomenon is of great interest and to be studied in future.

Fig. 3 .Fig. 2 .
Fig. 3. Physical BamHI, КрпІ and maps of the ManeNPV DNA.Letters correspond to the fragments in fig. 2 and the table.The arrows indicate the position and direction of the polyhedrin gene and position of the «break point* ).The submolar band of 4.8 kbp has been shown by hybridization (fig. 1, В, О to be present in the HindiII^REN pattern in major fraction fragments of the same size as hidden.These Hindlll submolar fragments sizes sum (4.8 kbp +1.3 kbp) corresponded to the size of the #mrf///-fragment hybridizing with the probes (6.3 kbp).We could not locate the EcoRI, Hindi11 and ^///-fragments directly on the ManeNPV physical map as the recognition sites for these restriction enzymes were not ordered in the full ManeNPV genome.However, the comparison of the full-size physical map and the mentioned £?cojR/-^-fragment map has shown that both BamHI-D and Kpnl-Gfragments are included in the £coff/-.4-fragmentgiving us a possibility to locate the break point on the £coK/-^-fragment map.To confirm our hypothesis concerning the existence of this point, we have analyzed the order of BgUI and Hindi I I sites inside the 2?caR/-;4-fragment.Our results have demonstrated that a possible break point is localized inside the 2.9 kpb-fragment dividing it into two parts of 2.0 and 0.9 kpb.The Hindi 11-fragment (6.1 kpb) is also a part of the £caR/-A-fragment and is divided by the break point into two fragments -1.3 and 4.8 kpb.Two minor £cojR/-fragments of 14.0 kpb and 10.0 kpb may be a result of the jEcoKZ-fragment (24.0 kpb) damage at the same point.All these results confirm the break point in the site located at a distance of approximately 21.0 kbp from the zero point on the ManeNPV physical map.To locate this site more precisely and to determine the distance from the PstI site, we have made the additional double REN digestion of the virus DNA using BamHI, Kpnl, Hindi 11, BgUI com bined with PstI, the REN products being hybridized with the labelled BamHI fragments (3.4 and 5.5 kpb) (data not shown).According to these results, the break point is situated at a distance less than 0.1 kbp downstream the PstI site.This fact explains the «absence» of submolar bands on the PstI REN pattern in fig. 1, A. The 2L0 kpb-fragment and the submolar fragment are almost of the same size, the difference being less than 0.1 kpb; so it is beyond the dif ferential ability of the REN analysis.The second band corresponding to the second minor component (less than 0Л kpb) is of a very low intensity and cannot be detected even by blot-hybridization ap proach.Having detected the break in the minor DNA fraction we are now to answer the question if such break is «naturally» present in a part of virus DNA molecules (before DNA isolation) or it appears as a result of the DNA molecule damage during its extrac tion and purification since some DNA molecules are damage-sensitive in a determined specific point.Our similar results with the virus DNA isolation using different protocols do not rule out this last possibility because each of them includes a phenol-chloroform extraction stage able to damage the DNA molecules.