Max D. Summers

Baculovirus structural polypeptides

Abstract The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica M NPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou M NPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis M NPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).

Electron microscopic observations on granulosis virus entry, uncoating and replication processes during infection of the midgut cells of Trichoplusia ni

A detailed ultrastructural study is presented showing the processes whereby intact granulosis virus particles invade and infect the midgut epithelial cells of Trichoplusia ni larvae by per os routes. Intact, enveloped nucleocapsids released from the proteinic crystals in the gut lumen apparently entered the columnar cells by interactions with the microvilli not yet defined and understood. The results of this study suggest that the viral envelope is lost at, or during, the entry process since only nonenveloped nucleocapsids were seen in cells shortly after infection. Phagocytosis of enveloped or naked nucleocapsids was not observed, nor were such virions observed in cytoplasmic vesicles shortly after infection. After entry, observations indicate that naked nucleocapsids interact end-on with the nuclear pore of susceptible cell nuclei to directly release viral contents into the nuclear region. Such observations as described above were frequently made shortly after introducing highly purified, occluded virions into the gut lumen. Virus progeny were observed in columnar cell nuclei 24 hours after virus entry. Certain aberrant forms of progeny nucleocapsids were also observed at that time. Assembled virions were not occluded in a proteinaceous crystal in midgut cells, but, for the most part, were released from the nuclei and caught up in vesicles which migrated toward the base of the cell, and into elements of the basement membrane; thus suggesting that virus progeny are released into the insect hemocoel by such processes. Plaque elements or structures were frequently seen on the surface of the virus-containing vesicles before and after release from the columnar cell into the hemocoel. Discussions are presented considering the source of the membranous structures associated with the virions.

Entry of an insect virus in vivo by fusion of viral envelope and microvillus membrane.

Abstract The entry of the Rachiplusia ou nuclear polyhedrosis virus into larval midgut columnar cells was observed in vivo. The nucleocapsids entered the microvilli after fusion of viral envelope with microvillus membrane. The subsequent stages of the invasion process appeared to be similar to that described earlier for other occluded rod-shaped insect viruses.

Ultrastructural studies on inclusion formation and virus occlusion in nuclear polyhedrosis and granulosis virus-infected cells of Trichoplusia ni (Hübner)

A description is given of the formation of viral and virus-free inclusions in nuclear polyhedrosis virus (NPV) infected cells of Trichoplusia ni. A relationship between “membrane-like” profiles, fibrous material, and inclusion bodies was shown which is believed to reveal a possible process by which protein subunits are assembled into a growing crystal. During development, the fibers were first observed forming between existing “membrane-like” profiles. The origin of the profiles is unknown, but they are of composite structure not previously shown in biological systems. Observations suggest that the profiles are related to or intimately associated with tubules having a unit membrane structure; these may possibly be derived from the nuclear membrane. The fibrous material associated with profiles also appears to be fused with the surface of growing crystals. A partly ordered structure exhibiting a line periodicity similar to that of completed crystals can be seen in the fibrous material. Similar fibrous material was also observed in the granulosis virus (GV) infected fat body cells and was often intimately associated with growing, but aberrant, inclusion formation. “Membrane-like” profiles were not observed in the GV replication cycle. Inclusions formed in the presence of large numbers of naked virus particles, but viral occlusion occurred only after acquisition of the outer membrane. Possible explanations for these observations are discussed.

Comparative studies of baculovirus granulins and polyhedrins.

Granulins and polyhedrins from five baculoviruses exhibit similar chemical and physical properties. Although similarities are demonstrated, it has been shown that each of the proteins is different and apparently specific to a given virus. The granulins and polyhedrins exhibit a major polypeptide component of an estimated molecular weight of 28,000. Alkaline protease activity has been detected in each preparation. N-terminal analyses reflect differences between granulins and polyhedrins studied: granulosis virus = Asx, and nuclear polyhedrosis virus = Glx. Two-dimensional high-voltage electrophoresis of highly purified granulin and polyhedrin preparations reveals relatedness as well as differences among the proteins as assessed by electrophoretic migration patterns.

Alkali-liberated granulosis virus of Trichoplusia ni: I. Density gradient purification of virus components and some of their in vitro chemical and physical properties

Abstract Trichoplusia ni capsules were purified by a combination of differential and ratezonal centrifugation on 50 to 80% sucrose gradients. The virus was released and purified from the inclusion body protein by exposure to weak alkali and subsequent centrifugation on 10 to 40% sucrose gradients. Virus preparations maintained in 0.01 m phosphate buffer (pH, 7.8) demonstrated 3 sedimenting forms on 10 to 40% sucrose gradients which were designated as A and B (with outer envelope) and Y (without outer envelope). The virus was unstable in 0.01 m phosphate buffer pH 7.8, and had a tendency to aggregate, and/or dissociate into the outer and inner membranes and a top fraction. The infection properties of each virus form and the top fraction were tested. Other buffers or additives did not change these phenomena. These virus forms and components were separated by a combination of sedimentations on 10 to 40% and 5 to 20% sucrose gradients and their ultrastructure confirmed by electron microscopy. Solutions containing virus forms A and B exhibited an E 260 E 280 of 1.26 and DNA and protein concentrations of 27.4 and 28.8 μg DNA/E260 and 105 and 109 μg protein/E260 for forms A and B, respectively. Preparations of the Y form contained 73.0 μg DNA/E260 and 258 μg protein/E260, but exhibited only 53% the ultraviolet extinction of virus forms A and B. Studies confirmed that the granulosis virus of Trichoplusia ni is ultrastructurally similar to other granuloses.

Observations on the morphogenesis and structure of a hemocytic poxvirus in the midge Chironomus attenuatus

An account is given of the ultrastructure of a poxvirus infecting larvae of a midge, Chironomus attenuatus. The virus appears to replicate only in hemocytes, and is of the occluded type. Immature virions are formed in association with an amorphous virogenic stroma, portions of which are sequestered into the developing particles. Further maturation of virions occurs either free in the cytoplasm or during (or after) occlusion within the crystalline matrix of polyhedra. A new intermediate in the development of cores from nucleoids is described. In addition, the presence of a unit membranelike structure in the poxvirus core is indicated. A possible nucleation site for the deposition of polyhedron protein is described.

Characterization of deoxyribonucleic acid isolated from the granulosis viruses of the cabbage looper, Trichoplusia ni and the fall armyworm, Spodoptera frugiperda.

Abstract Large, covalently closed circular deoxyribonucleic acid molecules have been isolated from the granulosis viruses that infect the cabbage looper, Trichoplusia ni, and the fall armyworm, Spodoptera frugiperda. As extracted from either virus, the nucleic acid is a mixture of double-stranded covalently closed circular molecules (about 30%), circular molecules (about 60%) with one or few nicks, and linear molecules (less than 10%). Centrifugation to equilibrium in cesium chloride gradients containing ethidium bromide shows the presence of three bands of DNA: a light band of DNA containing linear and relaxed circular DNAs, a heavy band of covalently closed DNA, and a third band of DNA intermediate to the light and heavy bands of DNA. Significantly, lyophilization of the occluded virus preparation before purification of the DNA does not allow the recovery of covalently closed DNA as compared to that recovered from nonlyophilized virus. Data herein suggest that lyophilization introduces, for the most part, a single-strand nick in the double-stranded molecule. A comparison of the sedimentation rates of the intact linear forms of the insect virus DNAs with those of T4 and T7 phage DNA standards on both neutral and alkaline sucrose gradients was used to estimate the molecular weight of the insect virus genomes. The genome from Spodoptera frugiperda granulosis virus is about 95 × 106 daltons and that from Trichoplusia ni granulosis virus is about 100 × 106 daltons. The base compositions of the virus and host DNAs were determined by thermal denaturation measurements.

Solubilization of Trichoplusia ni granulosis virus proteinic crystal: II. Ultrastructure

Abstract Various solvents (guanidine hydrochloride, normal propanol, urea, and sodium carbonate), previously demonstrated to solubilize Trichoplusia ni granulosis virus capsules, produced alterations which were characteristic for a given solvent. All solvents, with the exception of guanidine hydrochloride (G·HCl) caused longitudinal fragmentation of the proteinic crystal early in the solubilization process. G·HCl, in contrast, appeared to be capable of penetrating and producing small cavities within the crystal. Intact free virions were obtained only from carbonate-solubilized capsules. Capsules seemed to dissolve in carbonate by a sequential process. Evidence consistent with the occurrence of normally distributed differences in carbonate solubility among capsules is presented. The pattern of carbonate solubilization of the proteinic crystal and release of free virions most closely resembled the process in vivo as observed within the midgut lumen of larval Trichoplusia ni.

A mixed virus infection in midgut cells of Autographa californica and Trichoplusia ni larvae

Ultrastructural examination of the midguts of Autographa californica and Trichoplusia ni infected with a viral preparation obtained from Autographa nuclear polyhedrosis virus (AcMNPV)-infected insects revealed the presence of five morphologically distinct virus particles. Two virus particles were observed simultaneously in the nuclei, AcMNPV and an approximately 40-nm icosahedral unenveloped particle. Within the same cell two other viruses were observed in the cytoplasm, cytoplasmic polyhedrosis virus and a dense icosahedral unenveloped particle around 28 nm. A fifth particle resembling an icosahedral cytoplasmic deoxyribovirus was occasionally observed. Both the 40- and the 28-nm particles were observed to be occluded within polyhedra.