The Autographa californica Multiple Nucleopolyhedrovirusac54Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

ABSTRACTBaculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate theac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma,ac54ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasind-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected byac54deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly.IMPORTANCEBaculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins.

Identification of BV/ODV-C42, an Autographa californica Nucleopolyhedrovirus orf101-Encoded Structural Protein Detected in Infected-Cell Complexes with ODV-EC27 and p78/83

ABSTRACT orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.

Histopathology of cytoplasmic polyhedrosis virus (Reoviridae) infection in corn earworm, Helicoverpa zea (Boddie), larvae (Insecta: Lepidoptera: Noctuidae)

Histopathology of cytoplasmic polyhedrosis virus (CPV) infection in the larval midgut of the corn earworm, Helicoverpa (=Heliothis) zea (Boddie) is described. Small polyhedral inclusion bodies (PIB) are observed in columnar cells of the midgut 1 or 2 days after treatment with CPV. Virions are partially or completely occluded in a polyhedral matrix to form PIB at the periphery of the virogenic stroma. PIB are dodecahedral in shape with a hexagonal outline and measure from less than 1 to 4 μm in size. Virions measure about 50 nm in diameter. Microvilli of infected columnar cells are not affected until immediately prior to rupture of the cell. Some infected columnar cells ruptured to release PIB into the gut lumen 2 or 3 days after infection. PIB are also released into the lumen by extrusion of heavily infected columnar cells. Mitochondria and rough endoplasmic reticulum deteriorate as infection progresses, and in many cells with advanced infection, the nucleus is obscured. PIB are found in goblet cells 5 days after infection. Infected goblet cells degenerate to such an extent that only a few of the original microvillus-like cytoplasmic projections and cell organelles are left. The implications of CPV infection in integrated pest management of the corn earworm are discussed.

Virus-Like Particles in an Ultra-Oligotrophic Lake on Vancouver Island, British Columbia

Transmission electron microscopy (TEM) seasonal studies of concentrated water samples from Sproat Lake, Vancouver Island, British Columbia, revealed numerous polygonal virus-like particles of variable size (60–200 nm). These particles (ca. 107/mL) were either free-living or associated with host picoplankters. Negative staining of living samples provides clear evidence of early stages of phage–picoplankton interactions. These phages display a six-sided head (ca. 90 nm dia.) with a distinct appendage (ca. 200 nm) or striated tail (ca. 130 nm). Viruses with dense matrices, deprived of envelopes or occurring as empty shells were found in the marginal area of invaded cells. Morphological changes such as invagination of the photosynthetic lamellae with the appearance of 'virogenic stroma' or with disruption of the cell membrane and the cell wall are described. Comments on the possible functional significance of viral agents in the biology and ecology of host cells are presented.

Virus-like particles in the green alga Cylindrocapsa

Icosahedral, virus-like particles (VLPs) have been studied in attached germlings of Cylindrocapsa geminella Wolle after the settling of zoospores produced by a heat-shock treatment. About 26 h after zoospore attachment, 3–10% of the resulting one-celled germlings contained a massive concentration of VLPs filling a region at the basal (i.e., attached) end of each cell. This includes the area normally occupied by the nucleus which is completely lysed by the time the VLPs are first observed. VLPs were found only in single-celled germlings, although the samples included multicellular germlings. Organelle degeneration occurs in the infected germlings and this apparently leads to cell lysis. Mature VLPs generally appear six-sided in section and have a maximum diameter of 200–230 nm. They possess a finely fibrillar core surrounded by a multilayered, membranous coat. The varied appearance of the virogenic stroma is discussed as well as putative stages in viral assembly. Cultures of Cylindrocapsa are being maintained from which VLP-infected germlings can be routinely obtained. The possibility that these cultures may be carrying a latent virus infection which can be induced by heat shock is discussed.