Autographa Californica Multiple Nucleopolyhedrovirus P48 (Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus(AcMNPV) p48(ac103)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs). However,the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In Ac MNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.     

Studies on the pathology of a baculovirus in Aedes triseriatus

The pathology of a Baculovirus (BV) in Aedes triseriatus was studied. The virus infected the cardia, gastric caeca, and the entire stomach of larval midgut epithelium. The progression of the disease was similar to that of other Baculoviruses of the nuclear polyhedrosis virus (NPV) type. Rodshaped nucleocapsids were formed within a Feulgen-positive virogenic stroma and along the nuclear envelope. These nucleocapsids were enveloped by a membranous material and occluded randomly in small irregular and polyhedral proteinic inclusions. The disease differed from other BVs of the NPV type in that the small proteinic inclusions gradually coalesced as they grew, forming large fusiform inclusions.     

Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells.

Proper assembly of nucleocapsids of the baculovirus Autographa californica nuclear polyhedrosis virus is prevented by cytochalasin D, a drug that interferes with actin microfilament function. To investigate the involvement of microfilaments in A. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. Tetramethylrhodamine isothiocyanate-labeled phalloidin was used to label microfilaments, and monoclonal antibody was used to label p39, the major viral capsid protein. Three microfilament arrangements were found in infected cells. During uptake of virus, thick cables were formed. These were insensitive to cycloheximide, indicating that this configuration was a rearrangement of preexisting cellular actin mediated by a component of the viral inoculum. At the time of cell rounding and before viral DNA replication, ventral aggregates of actin were observed. These were sensitive to cycloheximide but not to aphidicolin, indicating that an early viral gene mediated this actin rearrangement. Ventral aggregates did not result from the rounding process itself. Uninfected cells prerounded with colchicine did not form ventral aggregates. Cells prerounded with colchicine and then infected did form aggregates. At the time of exponential production of progency virus, microfilaments were found in the nucleus surrounding the virogenic stroma. In this area (where nucleocapsid assembly is known to take place) microfilaments colocalized with p39. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis identified p39 among proteins retained on an f-actin affinity column. We postulate that microfilaments in the nucleus provide a scaffold to position capsids for proper assembly and filling with DNA.     

大菜粉蝶颗粒体病毒的形态发生及细胞病理的超微结构观察

本文叙述感染大菜粉蝶颗粒体病毒后,病虫脂肪体细胞超微结构的改变,大菜粉蝶感染后24小时,病虫脂肪体细胞开始出现明显的病变,整个病程是,在开始时细胞核内出现清晰区并出现病毒发生基质,核膜多点成套增生,其后核膜断裂,大量膜样结构聚集在病毒发生基质的周围,核衣壳大量产生,有一部分核衣壳从这些病毒发生基质四周的膜样结构碎片上获得套膜,荚膜蛋白沉积形成成熟的病毒荚膜,或称包含体;另一部分则排列在胞浆内的空泡边缘上;其余的核衣壳则从细胞边缘“芽突”而获得套膜,另外还描述环孔片层及线粒体改变。     

Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane

Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting nucleocapsids and the surface protein GP to filopodia, the sites of viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus-like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non-phosphorylatable form of VP40 (VP40(mut) ). VP40(mut) was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon-induced gene expression similarly to wild-type VP40. However, VP40(mut) was specifically impaired in its ability to recruit nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of nucleocapsids to the viral envelope.     

Virus envelope acquisition of nuclear polyhedrosis virus in vitro.

There have been several reports concerned with the replication and morphogenesis of insect baculoviruses during the past decade [1--7]. While there is general agreement as to the assembly of the virus on the basis of electron microscopic studies, there are still questions regarding the details of the replicative mechanisms, such as the acquisition of the virus envelope. Three possible ways have been proposed to describe envelope formation of nuclear polyhedrosis virus: (i) acquisition of budding through the nuclear membrane; (ii) acquisition of budding through the plasma membrane, and (iii) de novo formation within the nucleus. This paper briefly describes the observations made on the acquisition of a virus envelope by the cotton bollworm virus (Heliothis armigera), a nuclear polyhedrosis virus, in primary hemocyte cultures. Swirling hair-like clusters, hitherto unreported, were observed in association with virogenic stroma and nucleocapsids in the nuclei of infected cells. It is postulated that the formation of the hair-like structure may be involved in the process of envelopment of the virus.     

Autographa californica multiple nucleopolyhedrovirus 38K is a novel nucleocapsid protein that interacts with VP1054, VP39, VP80, and itself

It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusion-derived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.     

Proteomic Analysis of Mamestra Brassicae Nucleopolyhedrovirus Progeny Virions from Two Different Hosts

Mamestra brassicae nucleopolyhedrovirus (MabrNPV) has a wide host range replication in more than one insect species. In this study, a sequenced MabrNPV strain, MabrNPV-CTa, was used to perform proteomic analysis of both BVs and ODVs derived from two infected hosts: Helicoverpa armigera and Spodoptera exigua. A total of 82 and 39 viral proteins were identified in ODVs and BVs, respectively. And totally, 23 and 76 host proteins were identified as virion-associated with ODVs and BVs, respectively. The host proteins incorporated into the virus particles were mainly involved in cytoskeleton, signaling, vesicle trafficking, chaperone and metabolic systems. Some host proteins, such as actin, cyclophilin A and heat shock protein 70 would be important for viral replication. Several host proteins involved in immune response were also identified in BV, and a C-type lectin protein was firstly found to be associated with BV and its family members have been demonstrated to be involved in entry process of other viruses. This study facilitated the annotation of baculovirus genome, and would help us to understand baculovirus virion structure. Furthermore, the identification of host proteins associated with virions produced in vivo would facilitate investigations on the involvement of intriguing host proteins in virus replication.     

荨麻蛱蝶核型多角体病毒在病虫脂肪体中的形态发生

本文叙述了一种在高山草原地区较低温度下感染荨麻蛱蝶(Vanessa urticae)的核型多角体病毒的形态发生过程。荨麻蛱蝶幼虫经其病毒多角体感染5日后出现明显变化:细胞核膨大,核仁消失,核内出现清晰区及病毒发生基质。在病毒发生基质的周围,核衣壳大量产生。核衣壳是从这些病毒发生基质四周的模样结构碎片上获得套膜,装配成病毒粒子。随后病毒粒子逐渐进入多角体蛋白中,形成了成熟的单粒包埋型的多角体。观察结果表明,在较低温度下生长的荨麻蛱蝶NPV与在常温下生长的其它NPV有着类似的形态发生过程。     

Effects of the Epap granulovirus on its host,Epinotia aporema (Lepidoptera: Tortricidae)

The bean shoot borer, Epinotia aporema, is a major pest of soybeans in Argentina. Larvae of this pest are attacked by a granulovirus (EpapGV) that is the most important cause of sporadic epizootics in E. aporema populations. We studied the pathology of this virus in last-instar larvae using light and electron microscopy, and evaluated the effect of the disease on larval growth and development. EpapGV caused a polyorganotropic infection. No nucleocapsids were observed in the nuclei of infected cells prior to nuclear membrane disruption. Nevertheless, granulin was detected in the nucleus by immuno-gold staining, indicating that late gene expression occurred prior to nuclear membrane disruption. Establishment of the virogenic stroma led to complexes of continuous parallel convoluted membranous sheets. Nucleocapsids were enveloped in these areas to form virions, which were then occluded. Apparently as part of the cell-to-cell spread of infection, nucleocapsids were observed enclosed in large numbers within membrane-bound vesicles located between the cells and basal lamina. Larvae infected by EpapGV suffered a retardation of development and typically failed to pupate, but exhibited a weight increase greater than that of healthy E. aporema.     

Characterization of an extremely basic protein derived from granulosis virus nucleocapsids

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex.     

家蚕CPV感染离体细胞的电镜观察

本文报道了BmCPV感染家蚕细胞系后的电镜观察。病毒感染早期,细胞质内形成电子致密的病毒发生基质,由病毒发生基质形成BmCPV球状病毒粒子;病毒感染48小时后,多角体在病毒发生基质周围形成,大量的病毒粒子随机包埋在多角体内;病毒接种后96小时,多角体数目增多,其形状有三角,四角,五角及六角形,细胞质内充盈多角体致使细胞核被挤向细胞一侧并伴有形态的改变,受染细胞约为40％。     

Morphogenesis of nuclear polyhedrosis virus from Autographa californiea in a cell line from Mamestra brassicae (cabbage moth)

Summary Cultures of cell line IZD-MB 0503 from Mamestra brassicae were inoculated with nonoccluded nuclear polyhedrosis virus of Autographa californica (AcNOV). At 1 h postinoculation (p.i.) nucleocapsids were found in the cytoplasm near nuclear pores and within the nucleoplasm. Formation of virogenic stroma was observed at 7 h p.i. The first short empty capsids were seen at 10 h postinoculation (p.i.), followed by partially and completely filled nucleocapsids (11–12h p.i.) with most capsids filled at 12h and later. This suggests that nucleocapsid components, such as capsid and DNA-core, assemble in successive stages rather than simultaneously. Viral progeny, being released from the cells by budding, were observed at 13h p.i.Supported by Bundesministerium für Forschung und Technologie, Bonn, Kennzeichen PTB 8141     

A new pathogenic virus in the Caribbean spiny lobster Panulirus argus from the Florida Keys

A pathogenic virus was diagnosed from juvenile Caribbean spiny lobsters Panulirus argus from the Florida Keys. Moribund lobsters had characteristically milky hemolymph that did not clot. Altered hyalinocytes and semigranulocytes, but not granulocytes, were observed with light microscopy. Infected hemocytes had emarginated, condensed chromatin, hypertrophied nuclei and faint eosinophilic Cowdry-type-A inclusions. In some cases, infected cells were observed in soft connective tissues. With electron microscopy, unenveloped, nonoccluded, icosahedral virions (182 +/- 9 nm SD) were diffusely spread around the inner periphery of the nuclear envelope. Virions also occurred in loose aggregates in the cytoplasm or were free in the hemolymph. Assembly of the nucleocapsid occurred entirely within the nucleus of the infected cells. Within the virogenic stroma, blunt rod-like structures or whorls of electron-dense granular material were apparently associated with viral assembly. The prevalence of overt infections, defined as lethargic animals with milky hemolymph, ranged from 6 to 8% with certain foci reaching prevalences of 37%. The disease was transmissible to uninfected lobsters using inoculations of raw hemolymph from infected animals. Inoculated animals became moribund 5 to 7 d before dying and they began dying after 30 to 80 d post-exposure. The new virus is apparently widespread, infectious, and lethal to the Caribbean spiny lobster. Given the pathogenic nature of the virus, further characterization of the disease agent is warranted.     

Nonoccluded baculovirus- and filamentous virus-like particles in the spotted cucumber beetle,diabrotica undecimpunctata (coleoptera: chrysomelid)

Nonoccluded baculovirus-and filamentous virus-like particles were found in nuclei of hemocytes or midgut cells of field-collected spotted cucumber beetles. Each type of particle was associated with a different type of virogenic stroma containing various viral components similar to those referred to as capsid, nucleocapsid, viroplasm, and viral envelope in other known baculovirus infections. Nucleocapsids of the virus which occured only in hemocytes were rod-shaped particles approximately 230 nm long and 52 nm wide and were enveloped singly by a trilaminar unit membrane. Enveloped and partly enveloped particles appeared to be released from the nucleus to the cytoplasm by budding through the nuclear envelope acquiring additional membranes. The nucleocapsids of the virus which occurred only in nuclei of midgut cells were filamentous particles with an average diameter of 25 nm and variable length up to 2 μm. Some extremely long particles were bent almost 360° near the middle, resulting in a hairpin-like configuration. The particles were always enveloped singly. No particles budding through the nuclear envelope were observed.     

The FP25K Acts as a Negative Factor for the Infectivity of AcMNPV Budded Virus

Baculoviruses generally produce two progeny phenotypes—the budded virus (BV) and the occlusion-derived virus (ODV)—and the intricate mechanisms that regulate the temporal synthesis of the two phenotypes are critical for the virus replication cycle, which are far from being clearly understood. FP25K was reported to be responsible for the regulation of BV/ODV, and the mutations within result in a decrease of normal ODVs formation and an increase of BVs production. In this study, we demonstrated that the increase of BV titer in an fp25k knockout recombinant (fp25k-negative) was a result of higher infectivity of BVs rather than an increased production of BVs. The constitution of the major structural proteins and genome of parental and fp25k-negative BVs were analyzed. The results showed that the integrity of the majority of DNA packaged into the fp25k-negative BVs was intact; i.e., the genomic DNA of fp25k-negative BV had better transformation and transfection efficiency than that of the parental virus, indicating more intact genomes in the virions. Although the analysis of proteins associated with BVs revealed that more envelope protein GP64 were incorporated into the fp25k-negative BVs, subsequent experiments suggested that overexpression of GP64 did not improve the titer of BVs. Thus, we conclude that the main reason for higher infectivity of BVs is due to better genome integrity, which benefits from the deletion of fp25k resulting in increased stability of the genome and produce a higher proportion of infectious BVs. FP25K acts as a negative factor for the infectivity of BV.     

In rat dorsal root ganglion neurons,herpes simplex virus type 1 tegument forms in the cytoplasm of the cell body

The herpes simplex virus type 1 (HSV-1) tegument is the least understood component of the virion, and the mechanism of tegument assembly and incorporation into virions during viral egress has not yet been elucidated. In the present study, the addition of tegument proteins (VP13/14, VP16, VP22, and US9) and envelope glycoproteins (gD and gH) to herpes simplex virions in the cell body of rat dorsal root ganglion neurons was examined by immunoelectron microscopy. All tegument proteins were detected diffusely spread in the nucleus within 10 to 12 h and, at these times, nucleocapsids were observed budding from the nucleus. The majority (96%) of these nucleocapsids had no detectable label for tegument and glycoproteins despite the presence of tegument proteins in the nucleus and glycoproteins adjacent to the nuclear membrane. Immunolabeling for tegument proteins and glycoproteins was found abundantly in the cytoplasm of the cell body in multiple discrete vesicular areas: on unenveloped, enveloped, or partially enveloped capsids adjacent to these vesicles and in extracellular virions. These vesicles and intracytoplasmic and extracellular virions also labeled with Golgi markers, giantin, mannosidase II, and TGN38. Treatment with brefeldin A from 2 to 24 h postinfection markedly inhibited incorporation into virions of VP22 and US9 but to a lesser degree with VP16 and VP13/14. These results suggest that, in the cell body of neurons, most tegument proteins are incorporated into unenveloped nucleocapsids prior to envelopment in the Golgi and the trans-Golgi network. These findings give further support to the deenvelopment-reenvelopment hypothesis for viral egress. Finally, the addition of tegument proteins to unenveloped nucleocapsids in the cell body allows access to these unenveloped nucleocapsids to one of two pathways: egress through the cell body or transport into the axon.