Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy

Abstract

Virus assembly occurs in a complex environment and is dependent upon viral and cellular components being properly correlated in time and space. The simplicity of the flock house virus (FHV) capsid and the extensive structural, biochemical and genetic characterization of the virus make it an excellent system for studying in vivo virus assembly. The tetracysteine motif (CCPGCC), that induces fluorescence in bound biarsenical compounds (FlAsH and ReAsH), was genetically inserted in the coat protein, to visualize this gene product during virus infection. The small size of this modification when compared to those made by traditional fluorescent proteins minimizes disruption of the coat proteins numerous functions. ReAsH not only fluoresces when bound to the tetracysteine motif but also allows correlated electron microscopy (EM) of the same cell following photoconversion and osmium staining. These studies demonstrated that the coat protein was concentrated in discrete patches in the cell. High pressure freezing (HPF) followed by freeze substitution (FS) of infected cells showed that these patches were formed by virus particles in crystalline arrays. EM tomography (EMT) of the HPF/FS prepared samples showed that these arrays were proximal to highly modified mitochondria previously established to be the site of RNA replication. Two features of the mitochondrial modification are approximately 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle.

Fig. 1

Insertion of the tetracysteine motif into the FHV coat protein and expression in Drosophila cells. (A and B) A schematic representation of where the tetracysteine motif was inserted into coat protein for FHV_{tetracysteine 4–9} and FHV_{tetracysteine 268–269}. (C) DL1 cells were transfected with RNA1 and either RNA_{tetracysteine 4–9} or RNA2_{tetracysteine 268–269}, and 27 h post infection the cellular lysate was subjected to SDS-PAGE, which was probed with an anti FHV antibody. S2 cells were transfected with both RNA1 and RNA2_{tetracysteine 268–269} and labeled with ReAsH (red) at 21 h (D) and 27 h (E) post transfection. The mitochondria were labeled with an anti-biotin antibody (green) and the nucleus was labeled with DAPI (blue).

Fig. 2

EM of ReAsH labeled FHV_{tetracysteine 268–269}. S2 cells expressing FHVtetracysteine were labeled with ReAsH and a cell of interest was imaged with fluorescence microscopy (A). The cell was then subjected to photoconversion, which created an electron dense-precipitate around the FHV coat protein and was imaged with EM (B and C). The corresponding coat protein patches are numbered. The donut shape mitochondria are indicated by closed arrow while open arrows indicate the horseshoe shaped mitochondria. Scale bars: B – 5 µM, and C – 2 µM.

Fig. 3

Comparison of ultrastructure using chemical fixation versus high pressure freezing and freeze substitution. Drosophila cell were infected with FHV and at 12 h post infection the cells were prepared for EM using traditional chemical fixation (A and B) or HPF and FS (C and D). The samples prepared by HPF/FS had significant improvement in preservation compared to the samples prepared by traditional chemical fixation. With this improved quality of preservation the paracrystaline nature of the arrays is apparent and the virus particles are observed bound to the cell membrane (D). Scale bars: A and C – 1 µM, and B and D – 250 nm.

Fig. 4

EMT of FHV in Drosophila cells. Drosophila cells at 12 h post infection were subjected to HPF and FS, embedded, and cut into ~500 nm sections. Two orthogonal −60 to +60 degree tilt series in 2 degree increments were collected. Four sections approximately 120 nm apart and about 12 nm thick were extracted from the reconstructed volume (A, B, C, and D). Four mitochondria were observed in all the sections and are labeled M1, M2, M3, and M4. A closed arrow indicates the sections where the mitochondrial chambers are closed to the cytoplasm. Open arrows indicate the openings in the mitochondrial chambers. Scale bar: 500 nm.