In cultured cells the baculovirus P10 protein forms two independent intracellular structures that play separate roles in occlusion body maturation and their release by nuclear disintegration

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.

Fig 1. TEM image and SBFSEM reconstruction of baculovirus-infected cell structures.

(i) TEM image of a typical AcMNPV-infected TN-368 cell at 72 hpi highlights typical virus-infected cell structures (arrowed): virogenic stroma (VS); P10 (nuclear, n; cytoplasmic, c); electron dense spacers (EDS); and occlusion bodies (OB) as well as the cytoplasmic (CM) and nuclear membranes (NM). (ii) Whole cell imaging using SBFSEM on a typical AcMNPV-infected TN-368 cell at 72 hpi has been used to generate a 3D reconstruction of the structures shown in the TEM image.

Fig 2. P10 structures in AcMNPV-infected TN-368 cells from 24 to 96 hours post-infection.

(Ai) SEM images of resin embedded AcMNPV-infected TN-368 cells from the sample block face using the Gatan 3View system and a Zeiss Merlin compact field emission gun SEM at 24, 48, 72 and 96 hpi (see S2 Table for imaging parameters). (Aii) Whole cell 3D reconstruction (AMIRA) of nuclear and cytoplasmic P10 structures: nuclear P10 (dark blue), cytoplasmic P10 (light blue). (Aiii) P10 structures as in (Aii), plus reconstructed representations of virogenic stroma (yellow), occlusion bodies (dark grey) and electron dense spacers (red). (B) Estimate of P10 structure size using volumetric modelling data. (Bi) Datasets showing size range, mean measurements (nm) and (Bii) relative percentage volume of P10 structures compared to total cell (n = 2).

Fig 3. Surface generated images of AcMNPV-infected TN-368 cells at 72 hpi (i) and 96 hpi (ii) from SBF-SEM data sets.

Corresponding confocal microscopy images of AcMNPV-infected TN-368 cells at 72 hpi (i–ii) and 96 hpi (iii-iv). Black arrows (SBFSEM) and white arrows (confocal) highlight P10 (green) features. Scale bar 3 μm (SBF-SEM) and 20 μm (confocal). Infected cells were fixed at 96 hpi and stained by indirect immunofluorescence using an anti-P10 antibody. A secondary antibody conjugated to an Alexa-fluor 488 was used to visualise P10 structures.

Fig 4. Nucleotide sequence of p10 promoter leader sequence.

(A) Indicates site for a series of deletions made to the promoter sequence from *transcription initiation (mRNA CAP) and location of relevant restriction digestion sites. (B) Displays nucleotide sequence of PCR fragments made using a two-step PCR to generate a series of promoter deletions as indicated in (A). The black arrows indicate binding of primer set for the first round PCR, the dotted blue line was use for the second round PCR and was used in combination with the forward primer upstream of the p10 transcription start. The p10 coding region starts at the ATG start codon as indicated by the open arrow and p10 coding region is highlighted as grey text and underlined with a black line in (A).

Fig 5. Effect of modulation of p10 expression on P10 structure formation.

(A) Infected TN-368 cells were harvested at 96 hpi and total protein fractionated in a 15% (w/v) SDS-PAGE for either Coomassie staining (i) or western blot analysis (ii) with P10- or cathepsin-specific antibodies detected using an alkaline phosphatase-conjugated secondary antibody. Lanes 1–9 (Aii) correlate to sample names at top (Aii). Arrows show expected sizes of P10 (black) and cathepsin (white). Ladder size (kDa) is indicated, left. (B) Bar graph showing mean relative P10 band density for AcMNPV, Ac_P10^Rescue Ac_P10^prl-4, Ac_P10^prl-8, Ac_P10^prl-12, Ac_P10^prl-16 and Ac_P10^prl-20 from a series of western blots (n = 3) using Image J. (C) Box and whisker plot showing enumeration of released occlusion bodies of TN-368 cells infected with AcMNPV or recombinant viruses (5 MOI) at 7 dpi (n = 10 f.o.v images per virus). (D) TN-368 cells were infected with AcMNPV or recombinant viruses (5 MOI) in 35 mm dishes and incubated at 28°C. The cells were imaged using a light microscope Zeiss Axiovert 135 at 7 dpi to show cell morphology and lysis (100X). Scale bar is 20 μm. (E) Confocal microscopy of TN-368 cells infected with Ac_P10^Rescue, Ac_P10^prl-4 and Ac_P10^prl-20 to observe P10 structures at 96hpi (green). Scale bar 20μm. Abbreviation: c cytoplasm, n nucleus, f.o.v field of view.

Fig 6. SEM and TEM images, and SBF-SEM 3D models, visualise the association between electron dense spacers, P10 and occlusion bodies in AcMNPV-infected TN-368 cells at 24, 48, 72 and 96 hpi.

A) SEM (i, ii, iv, v) and TEM images (iii, vi) of occlusion bodies (OBs) from Ac_p10^Rescue and AcΔp10-infected TN-368 cells. OBs from AcΔp10-infected cells are pitted with a rough surface as indicated by arrow (iv, v) and are absent of a closely associated EDS as observed for Ac_p10^Rescue (vi). Scale Bar (i) 2 μm, (ii, iv, v) 1 μm (iii, vi) 500nm. B) SBF-SEM images (i) and surface generated models (ii, iii) from AcMNPV-infected TN-368 cells highlighting P10, EDS (black arrow) and OBs. Nuclear P10 (blue) is modelled as 24, 48, 72 and 96 hpi with EDS (red) and OBs (black). Detailed observations of OBs (iii) show the progressive encapsulation process of the OBs. Abbreviations: EDS electron dense spacers, OB occlusion bodies, C cytoplasm, N nucleus. Black arrows indicate EDS. Scale bar = 3 μm.

Fig 7. Structural model for the role of cytoplasmic P10 in nuclear lysis.

P10 (blue) presents as a thin filamentous structure that develop into a complex network of P10 tubules that wrap around the nucleus forming a peri-nuclear cage-like structure. The cage-like structure continue to remodel over time and by 96 hpi most P10 has coalesced into a polarised mass. This final remodelling is associated with loss of nuclear integrity, resulting in nuclear lysis and release of occlusion bodies.