Abstract
P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.
Highlights
The Baculoviridae is a diverse family of insect-specific viruses that contain circular, supercoiled dsDNA genomes ranging from 80 to 180 kilobase pairs [1, 2]
High-resolution 3D electron microscopy has revealed the complexity of structures formed by P10, a small 10kDa protein that accumulates to very high levels in baculovirus-infected cells
We demonstrate the formation and presence of two distinct, possibly unique, P10 structures that account for the diverse roles associated with this small protein
Summary
The Baculoviridae is a diverse family of insect-specific viruses that contain circular, supercoiled dsDNA genomes ranging from 80 to 180 kilobase pairs (kbp) [1, 2]. The expression system has exploited the highly efficient promoters of the p10 and polyhedrin (polh) genes, both of which have been found to be non-essential for the propagation of the virus in cell culture [12,13,14]. Over the course of a baculovirus infection, whether in larval tissue or in cultured insect cells, cell nuclei become enlarged and, in the very-late stages, become packed with the paracrystalline OBs that are readily visible under the light microscope [15]. Nuclei disintegrate releasing OBs or polyhedra into the liquefied larval remains or cell culture medium [16, 17]. The major component of OBs is the 30 kDa polyhedrin protein that is produced under control of the very late, hyper-expressed polh promoter [18]. It has been shown that in the absence of P10 or pp, OBs do not fully mature leaving a rough and pitted surface [22]
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