T h e first chemical analyses of polyhedra were carried out by Bolle (1873, 1893). He found that polyhedra consist of protein and contain no lipids. In evaluating the results of chemical analyses of polyhedra, one should realize that polyhedra consist of essentially two components:
the polyhedron protein which constitutes about 95 percent of the total weight, and the virus particles which amount to 5 percent. All analyses of total polyhedra are, therefore, analyses of polyhedral protein plus the virus particles. T h e most important chemical analyses of polyhedra and purified polyhedron protein are summarized in T a b l e I I . This T a b l e shows that the nitrogen content of polyhedra and the purified polyhedron-protein preparations from different hosts does not vary much and is about 14 to 15 percent. However, the phosphorus content of polyhedra varies with the preparation of purified polyhedron protein.
It is doubtful whether the small amount of phosphorus (about 0.05 per
cent) that cannot be removed belongs indeed to the protein molecules, or whether it is only adsorbed phosphorus. There is a difference between the phosphorus content of polyhedra and of polyhedron protein of about 50 to 60 percent. This difference is due to free dialyzable phosphate which is liberated from the polyhedra by the alkaline treatment (Desnu-elle et al, 1943; Bergold, 1947). Faulkner (1962) found R N A in B. mori polyhedra. T h e base composition (expressed as moles percent of total bases) of all extractable R N A was 16.2 guanine, 36.8 adenine, 8.1 cytosine, and 38.9 uracil after phenol treatment, and 18.3, 45.7, 8.2, and 27.8 after perchloric acid treatment of polyhedra. T h e function of this R N A is not known. T h e base composition of this R N A differs from that of the host insect where 29 guanine, 24 adenine, 25 cytosine, and 22 uracil were found by Eto (1955). An investigation of the metal content has revealed that there is only 0.005 percent iron in B. mori polyhedra and polyhedron protein, and 0.083 percent magnesium in polyhedra, but not in poly
hedron protein (Holoway and Bergold, 1953, 1955).
T h e first investigations of the amino acid content of B. mori poly
hedra were carried out by Desnuelle et al. (1943) and Desnuelle and Chang (1943). They found (expressed as percent of protein nitrogen), 0.4 cysteine and cystine, 4.6 alanine, 5.2 tyrosine, 4.7 histidine, 12.1 arginine, 3.8 phenylalanine, 3.0 tryptophan, 6.4 ammonia, and 3.8 humin nitrogen. T h e composition of amino acids of purified polyhedron protein from different insect viruses was investigated extensively by Wellington
436 GERNOT Η. BERGOLD
(1951, 1954). T h e results, summarized in T a b l e I I I , reveal that all analyzed inclusion-body proteins have a very similar amino acid composi
tion independent of whether the polyhedra were derived from a lepi-dopteron or a hymenopteron (N. sertifer). A comparison of any two of the proteins investigated show significant differences (P < 0.01) in the quantity of amino acids. No qualitative differences in amino acids were
T A B L E I I
COMPOSITION IN Bombyx mori OF INCLUSION BODIES, INCLUSION-BODY PROTEIN, AND VIRUS«
Substance Polyhedra
a Values are given as percentages.
& Bergold (1947).
* Bergold and Wellington (1954).
d Desnuelle et al. (1943).
* Glaser and Stanley (1943).
/ Ikeda (1946, 1951).
0 Yagi et al. (1951).
Ä Manunta (1940).
TABLE III AMINO ACID COMPOSITION OF INCLUSION-BODY PROTEINS^ δ Polyhedron type Porthetria Malacosoma Malacosoma Bombyx Colias Neodiprion Amino acid dispar americanum disstria mori eurytheme sertifer Number of hydrolyzates analyzed 3 2 2 5 2 2 Aspartic acid 11.1 ± 0.35c 13.5 13.6 12.5 ± 0.29 13.3 12.4 Glutamic acid 13.2 ± 0.30 12.4 11.7 12.7 ± 0.30 12.8 13.5 Histidine 3.7 ± 0.44 2.8 2.2 2.8 ± 0.18 2.8 3.6 Lysine 8.5 ± 0.29 9.4 6.3 10.5 ± 0.25 8.7 6.6 Arginine 9.2 ± 0.26 9.7 9.4 6.8 ± 0.33 10.1 10.3 Glycine 3.1 ± 0.12 2.7 2.8 3.3 ± 0.09 3.0 3.6 Alanine 3.3 ± 0.26 2.9 2.8 2.9 ± 0.05 2.8 2.6 Valine 6.0 ± 0.24 6.9 5.8 5.7 ± 0.16 7.0 5.7 Leucine and/or isoleucine 13.0 ± 0.46 14.3 13.4 13.3 ± 0.20 13.2 14.7 Proline 4.9 8.2 6.0 6.0 8.3 5.2 Phenylalanine 7.5 ± 0.66 7.0 7.1 6.9 ± 0.20 6.4 9.5 Tyrosine 10.0 ± 0.23 9.6 10.1 10.9 ± 0.21 7.6 5.3 Serine 3.8 ± 0.07 3.6 4.3 3.3 ± 0.07 3.6 3.6 Threonine 4.7 ± 0.15 3.1 4.6 3.8 ± 0.14 3.0 4.9 Cysteine and /or cystine 1.1 ± 0.00 1.3 1.1 0.6 ± 0.08 1.3 1.6 Methionine 1.7 ±0.18 2.4 2.3 3.0 ± 0.09 2.3 3.3 Total 104.8 109.8 103.5 105.0 106.2 106.4 Total calculated as amino acid residues per 100 gm protein 91.2 95.2 89.7 90.5 90.60 92.1 a From Wellington (1954). δ Estimated by paper chromatography of acid hydrolyzates, and expressed as grams amino acid per 100 gm material analyzed. Tryptophan, also present in these proteins, was not estimated. ο Mean value and its standard error.
438 GERNOT Η. BERGOLD
found between the membranes of P. dispar polyhedra and the poly
hedron protein (Wyatt, 1950).
It was shown by Desnuelle and Chang (1945) that treatment of B. mori polyhedra with anhydrous methanol and dried hydrogen chloride gas converts all carboxyl groups to methyl esters (111 groups), and the basic groups to hydrochlorides (67 groups).
VI. CHEMICAL COMPOSITION OF VIRUS PARTICLES
T h e first report that polyhedra (of P. dispar) give a positive Feulgen reaction was published by Breindl and Jirovec (1936). W e know, of course, that this reaction is due to the DNA content of the enclosed virus particles. Alkaline solutions of polyhedra have ultraviolet-absorp
tion spectra; these spectra suggest also the presence of nucleic acid (Dannenberg, cited in Bergold and Schramm, 1942). This was confirmed by chemical tests, but no uronic acid or free carbohydrates could be detected (Tarasevich, 1946). T h e first quantitative determinations are reported by Gratia et al. (1945), who found 0.48 percent DNA, but no RNA, in B. mori polyhedra. An analysis of purified virus particles revealed 13 percent DNA in virus particles of B. mori, and 16 percent in those of P. dispar (Bergold, 1947; Bergold and Pister, 1948). Careful analy
ses of purified virus suspensions of different insects showed no trace of RNA (Wyatt, 1952a, b). A chemical reinvestigation of very highly purified B. mori particles indicated that they contain about 7.9 percent DNA and 0.915 percent phosphorus, of which, however, only 87 percent is found in the DNA (Bergold and Wellington, 1954). T h e ratio of DNA basis to total phosphorus is in good agreement with this (Wyatt, 1952b). T h e remain
ing 13 percent phosphorus may originate from the surrounding virus membranes, which contain about 0.45 percent phosphorus. In the virus membrane, there is only about half as much nondialyzable phosphorus as there is in the virus. Nothing is known as to the kind of bond involved. Krieg (1956) found 9 percent DNA, but no RNA, in a purified preparation of the nuclear virus particles from A porta crataegi (Lin
naeus). Analyses of the nitrogen and phosphorus content of different virus particles and virus membranes are summarized in Tables I I and I I I . These tables indicate that virus membranes obtained by alkaline treatment and separation from the virus particles by ultracentrifugation have less nitrogen (12.5 percent) than have the virus particles (13.9 per
cent) (Bergold and Wellington, 1954).
Manunta, in 1940, was the first to report the presence of purine bases in B. mori polyhedra. T h e results of intensive investigations (Smith and Wyatt, 1951; Wyatt, 1952b) of the bases of several insect viruses are summarized in T a b l e IV. This table shows that nuclear insect viruses
TABLE IV PURINE AND PYRIMIDINE COMPOSITION OF DNA OF INSECT VIRUSES* Percentage of total Ρ Host order and Number of Moles per 100 moles total basesc accounted Adenine + thymine species family analyses** Adenine Thymine Guanine Cytosine for guanine -f- cytosine Lepidoptera Porthetria dispar Lymantriidae 4 21.2 20.05 30.5 28.25 92 0.71 Lymancha monacha Lymantriidae 1 24.6 23.8 26.8 24.7 — 0.94 Choristoneura fumiferana Tortricidae 3 24.8 24.0 26.7 24.5 86 0.95 Ptychopoda seriata Geometridae 2 26.7 25.7 24.4 23.2 87 1.10 Malacosoma americanum Lasiocampidae 3 29.2 28.0 22.5 20.2 93 1.34 M. disstria Lasiocampidae 3 29.2 28.5 21.9 20.3 86 1.36 Bombyx mori Bombycidae 3 29.3 28.0 22.5 20.2 88 1.34 Colias eurytheme Pieridae 4 29.9 27.6 22.4 20.1 90 1.35 Hymenoptera Neodiprion sertifer Tenthredinidae 2 32.3 30.3 19.5 17.8 — 1.67 a From Wyatt (1952). & Independent analyses performed on different preparations of virus. c Mean value and its standard error.
440 GERNOT Η. BERGOLD
contain, as would be expected due to the lack of RNA, only the purines, adenine and guanine, and the pyrimidines, cytosine and thymine, but no 5-methylcytosine or uracil. T h e ratios of adenine to thymine and of guanine to cytosine are about 1.0 and are almost constant, but the ratio of adenine + thymine to guanine -f- cytosine varies. T h e differences of the ratios in viruses from different insects occur in steps (0.7; 0 . 9 5 -1.10; 1.34-1.36; 1.67; and 1.87), forming groups of similar or identical ratios. T h e ratios of closely related hosts (Malacosoma disstria Hübner, Malacosoma americanum (Fabricius), and Choristoneura fumiferana) have similar ratios as one would expect. Even viruses of widely sepa
rated lepidopterous hosts and a nuclear virus from a hymenopteron (N.
sertifer) have similar A T / G C ratios. T h i s could be interpreted that not all DNA carried genetic information.
A search for metals by spectographic and analytical methods has revealed that B. mori virus particles contain only iron (0.015 percent) and magnesium (0.03 percent) (Holoway and Bergold, 1953, 1955).
T h e significance of such low quantities may be doubtful, but it is sur
prising that there are three times as much iron and four times as much magnesium in the virus particles as in the surrounding polyhedron protein.
T h e amino acid composition of several nuclear viruses was thoroughly investigated by Wellington (1951, 1954) (see T a b l e V). One can learn from this table that the five different nuclear viruses have all a similar pattern of amino acid composition, which, however, differs greatly from the pattern of the surrounding inclusion-body proteins. T h e virus, for instance, contains proportionally twice as much arginine, but only half as much lysine and tyrosine, and has a greater variability than the poly
hedron protein. Between any two viruses, there are significant differences in the amino acid composition which appear to be parallel to the taxonomic classification rather than to the grouping based on nucleic acid analyses. For instance, virus particles of B. mori, M. disstria, and M. americanum are similar in their nucleic acid, but not in their amino acid composition. T h e virus of P. dispar appears to be different from all other viruses in nucleic acid composition as well as in morphological characteristics.
Another investigation (Bergold and Wellington, 1954) has revealed that the amino acid composition of the virus membranes and that of the viruses is quite different; the virus membranes contain more aspartic acid and much less arginine than the virus. T h e amino acid composition of B. mori virus particles, the virus membranes, and the polyhedron protein are summarized in T a b l e V I . This table shows clearly differences between the three. Furthermore, it was found that the virus membranes
13. NATURE OF NUCLEAR-POLYHEDROSIS VIRUSES 441
Polyhedron type
Porthetria Malacosoma Malacosoma Bombyx
Amino acid dispar americanum disstria mori
Number of hydrolyzates
analyzed 2 2 1 2
Aspartic acid 12.0 11.0 11.0 13.3
Glutamic acid 8.5 9.3 10.0 6.9
Histidine 0.9 1.5 1.1 0.7
Lysine 2.1 3.5 3.5 3.3
Arginine 19.8 16.1 15.5 11.1
Glycine 6.1 4.0 4.9 8.1
Alanine 5.9 3.9 3.6 4.5
Valine 4.4 3.6 3.1 3.8
Leucine and/or isoleucine 11.0 11.8 12.7 11.2
Proline 6.4 6.3 5.2 4.9
Phenylalanine 4.5 4.7 5.5 4.6
Tyrosine 2.8 4.8 6.1 6.2
Serine 9.4 10.5 9.0 8.5
Threonine 4.4 6.0 6.4 9.6
Cysteine and/or cystine 0.9 0.8 0.6 0.7
Methionine 1.0 2.4 2.4 2.8
Grams amino acid recovered
per 100 gm virus 57.5 87.1 83.7 75.9
a From Wellington (1954).
δ Expressed as percentage of total recovered amino acids. T h e presence or absence of tryptophan could not be established.
and humin were applied, these figures might be in reality even higher.
A difference in the amount of amino acid Ν and of total Ν is attributa
ble to nitrogenous compounds and decomposition products not recov
ered. Because of the interference of DNA in the color reaction, the tryptophan content of the virus could not be determined. On the basis of total Ν content and the Ν content and weight of the recovered amino acids, one can conclude that the virus membranes consist of about 80 percent, and the virus of about 85 percent, nitrogenous compounds. Only about 1.3 and 0.2 percent lipids could be extracted with boiling petrol dissolve completely in 0.01 Μ alkali, but not in alcohol, ether, or cold HCl of any concentration. Only 57 percent of the weight of the virus membranes could be accounted for as amino acids, 64.4 percent of that of the virus, and 94.5 percent of that of the polyhedra. T h e analyses account, on a nitrogen basis, for 94.4 percent of the Ν in the membranes, 86.1 percent of the Ν in the virus, and 95.2 percent of the Ν in the polyhedron protein. Because no corrections for ammonia, tryptophan,
T A B L E V
AMINO ACID CONTENT OF INSECT VIRUSES^ &
442 GERNOT Η. BERGOLD
ether from virus membranes and from the virus particles. An extraction with a mixture of chloroform and methanol (4:1, 1 hour at 50° to 55°G, and additionally overnight at 20°C) of the virus particles yielded a maximum of about 7.5 percent extracted material from the virus particle.
About half of this 7.5 percent could be accounted for as total lipids, and 1.2 percent of carbohydrates could be found in the virus with the an-throne reaction (Bergold and Wellington, 1954). Taking all these analyses
T A B L E VI
AMINO ACID COMPOSITION OF THE VIRUS MEMBRANES, VIRUS, AND POLYHEDRON PROTEIN ON A NITROGEN BASIS»
Amino acid
Amino acid N2 as percent of sample N2
Amino acid Membranes Virus Polyhedron
Cystine and/or cysteine 0.6 0.4 0.5
α From Bergold and Wellington (1954).
& Corrected for glycine produced by hydrolysis of nucleic acid, c Determined by the method of Gordon and Mitchell (1949).
into account, one must conclude that about 9 percent of the weight of the virus, and a higher percentage of virus membranes, is still not accounted for.
Protein-free DNA and DNA-free protein were first isolated by Ger
shenson (1956b, c) with NaCl from A. pernyi polyhedra. However, neither preparation was significantly infectious when injected singly or in succession. By mixing the DNA and the protein preparations and leaving them together for 7 hours before injection, a 40 percent mor
tality resulted when about 1 mg DNA and 5 mg protein per pupa were
13. NATURE OF NUCLEAR-POLYHEDROSIS VIRUSES 443 injected. DNA was isolated from purified B. mori virus particles using
^-aminosalicylate and phenol by Bergold (1958b, c ) . T h e very viscous DNA preparation had an 52 0 of about 14.5 Svedbergs, but was contam
inated with some protein. This protein, however, was serologically not related to virus proteins. T h e infectivity of this DNA preparation
(0.1 mg per B. mori larva) was only 0.0001 percent that of the corre
sponding amount of untreated DNA still contained in the intact virus particle.
A most interesting investigation was recently reported by Gershen
son et al. (1960; 1961). With a modified phenol method, he isolated RNA from B. mori infected with nuclear-polyhedrosis virus (containing exclusively D N A ) , and found this R N A to cause nuclear polyhedrosis.
This infectious R N A was highly sensitive to ribonuclease, but not at all to deoxyribonuclease, treatment. It lost its infectivity standing at room temperature for 24 hours. T h e above investigation makes it highly probable that R N A synthesized in cells infected with the DNA-containing nuclear-polyhedrosis virus is infectious, causing nuclear poly
hedrosis. This result would be of utmost importance if found to be a general phenomenon; it would confirm Stent's assumption (1958) that RNA is controlling some stages of the multiplication of DNA-contain-ing viruses.
A catalase, as well as a proteinase, was reported to be present in alkaline solutions of B. mori polyhedra (Yamafuji et al., 1941, 1957).
In other investigations, no peptidase or phosphatase could be found (Duspiva and Bergold, 1942), and similarly, no evidence for the pres
ence of lipase, lecithinase, hexosediphosphatase, nuclease, amylase, car
boxylase, phenyloxidase, dehydrogenase, catalase, or protease could be obtained in polyhedron solutions (Kuzin and Krzhevova, 1948). T h e presence of a deoxyribonuclease reported by Yamafuji (1956) and Ya
mafuji et al. (1957) in alkaline solutions of B. mori polyhedra could not be confirmed by reinvestigation. No significant amount of deoxyri
bonuclease is present in suspensions of polyhedron solutions and puri
fied virus particles (Faulkner and Bergold, 1957).
Watanabe (1941a) reported that B. mori virus obtained by Berkefeld filtration is adsorbed onto kaolin (negatively charged) and onto alum
inum hydroxide (positively charged) within a pH range of 5.0 to 6.0.
It can be eluted with 0.2 Μ phosphate buffer of a pH of 9.0 to 9.5, with a considerable loss of infectivity. In the electrical field, the virus migrates toward the cathode at a pH of 4.5 to 5.7 and to the anode between pH 6.9 and 10.0 (negatively charged), but it does not move between pH 5.8 and 6.3 (Watanabe, 1941b). Aizawa (1952) found that the isoelectric point of the virus is at pH 6.0 to 6.2.
444 GERNOT Η. BERGOLD
V I I . SEROLOGICAL PROPERTIES AND RELATIONSHIP OF NUCLEAR-POLYHEDRON PROTEINS, NUCLEAR-POLYHEDROSIS VIRUSES,
AND INSECT HOSTS
T h e first serological investigations of polyhedra and alkaline solu
tions of them were carried out by Aoki and Chigasaki (1921). They found that B. mori polyhedra and solutions of them have good anti
genic properties, and are serologically not related to healthy B. mori hemolymph. Many serological investigations have been carried out since then, which are summarized in the following section.