Insights from the Infection Cycle of VSV-ΔG-Spike Virus

Abstract

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3-4 h post infection (PI), and accumulates as the infection proceeds. By 10-24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.

Keywords: RNA virus; electron tomography; spike protein; transmission electron microscopy; vesicular stomatitis virus; viral factories.

Figure 1

(A) Generation of rabbit serum antibodies. Rabbits were immunized with wild type-VSV (day 0) and at day 15 post prime injection, the rabbits were boosted with a similar dose subcutaneously. Serum was collected 15 days post boost. (B) Cells were either mock or VSV-S infected and then processed for immuno-florescence microscopy with 1:200 diluted rabbit serum antibodies. VSV proteins (Red) positive signal was visible in cytoplasm of infected cells, whereas in non-infected cells no staining of VSV proteins was seen. Nuclei of the cells are marked with blue color (DAPI). Scale bar is 20 µm.

Figure 2

TEM images of thin sections prepared from Vero E6 cells infected with VSV-S. The cells were infected with the virus at an MOI of 1 and then processed by chemical fixation at the indicated time points post infection (PI). (A) Non-infected cells show no virus particles in the cytoplasm. The nucleus (Nu) of the cell is visible with surrounding mitochondria. (B) Six hours PI, a typical cell exhibits no virus particles in the cytoplasm. Membrane cisternae (white arrows), ribosomes, and multi lamellar bodies (MLBs) are frequently seen. (C–F) At 8–23 h PI, “bullet like” structures appear in vacuoles (panels (C,D), red arrows) in cytoplasmic MLBs (panel (E), red arrow), or adjacent to the outer leaflet of the plasma membrane (panel (F), red arrows). In panels (C,D), insets show high magnification of delineated areas. M—mitochondria, Nu—nucleus, MLB—multi lamellar bodies.

Figure 3

RT real-time PCR analysis results of two viral genes, nucleocapsid (N) and spike (S). Initial detection at 3–4 h post infection (PI) can be seen. As infection progresses, the reduction of Ct values suggests active viral RNA replication. The limit of detection for detection of S gene was 10 pfu/mL, and 100 pfu/mL for N gene. At 1–2 h PI, viral RNA was not detected due to low concentration below the limit of detection.

Figure 4

VSV-S viral factories (VF) are part of a complex network composed of cellular membranes and multi lamellar bodies (MLB). (A,B) Two tomographic slices from two different tomograms of VSV-S infected cells at 16 (A) and 23 (B) hours post infection (PI). (C–F) High magnification views of the VF 1–4 shown in panels (A,B), respectively. VSV-S VF are located in multi lamellar structures with adjacent host membrane cisternae (blue arrows and inset that shows high magnification of delineated area with host ribosomes on a membrane cisterna). The VF consist of empty particles (green arrows) as well as viruses already packed with their genome (red arrows) or other virus morphologies with partial density inside (yellow arrows). White arrows point to “onion like” membrane stacks or linear membrane sheets that are part of the MLB. (G,H) 3D volume rendering of the tomogram in panel (A) showing the MLB “onion like” composition (light blue), stack of membrane sheets (green) and adjacent membrane cisternae (light blue). Various virus morphologies of VSV-S particles depicted in yellow. Scale bars in panels (A,B) are 200 nm and (C–F) 100 nm. Thickness of tomographic slices are 8.5 nm (A), 6.8 nm (B).

Figure 5

VSV-S genome replication occurs in discrete foci in the cytoplasm where VSV-S proteins accumulate. (A–I) Cells were either mock or VSV-S infected at the indicated time points and then processed by immuno-labeling with anti-VSV (A,D,G) and anti-dsRNA (B,E,H) antibodies. (A–C) Non-infected cells did not show any labeling of VSV proteins or dsRNA. (D–F) A representative image of cells infected for 4 h exhibiting no VSV proteins labeling. Scarce labeling of dsRNA in the cytoplasm is noticed. (G–I) At 10 h post infection, part of the cells were positive both for VSV proteins and dsRNA at distinct sites in the cytoplasm. (C,F,I) Merge channels of dsRNA (red), VSV proteins (green) and DAPI (blue) for nuclei staining. Scale bars are 20 µm. Objective lens X63.

Figure 6

VSV-S proteins accumulate at distinct sites in the cytoplasm where ER is absent. (A–F) Cells were either mock or VSV-S infected for 10 h and then processed for immuno-labeling with anti-VSV antibodies (A,D) or stained for ER detection (B,E). (A–C) In non-infected cells the ER (red) accumulates around the nuclei. (D–F) At 10 h PI, VSV-S proteins (green) accumulate in regions where ER is absent. (C,F) Merge channels of both ER, VSV proteins and DAPI (blue) for nuclei staining. Scale bars are 20 µm. Objective lens X63.

Figure 7

VSV-S particles are found in vacuoles after release from virus factories in multi lamellar bodies (MLB). (A–F) Sequential tomographic slices of VSV-S infected cells at 16 h (A–C) and 24 h post infection (PI) (D–F). Red arrows point toward several VSV-S particles inside a vacuole connected to MLB containing VF. Insets are high magnifications of delineated areas. Tomographic slices thicknesses are 13 nm (A–C) 4.3 nm (D–F). Scale bars are 200 nm.

Figure 8

Release of VSV-S particles occurs after fusion of the vacuole membrane and the plasma membrane. (A–C) Sequential tomographic slices from a tomogram of 24 h post infection VSV-S infected cell. Red arrow points toward a VSV-S particle in a membrane vacuole (A). As the volume changes the vacuole becomes narrower ((B), red arrow) and at a certain point in the volume, is connected to a protrusion in the plasma membrane ((C), red arrow). (D–F) 3D surface rendering of the tomogram in panel (A). A large vacuole is depicted (blue color) with numerous virus particles decorated with probable spike proteins (yellow). A vacuole containing an individual virus particle is located nearby and is connected to the plasma membrane (blue) through a narrow membrane neck (red arrow). Tomographic slice thicknesses are 7 nm. Scale bars in panels (A–C) are 100 nm.

Figure 9

A model illustrating key processes in the infection cycle of VSV-S. Assembly and genome packaging of VSV-S particles is carried out in viral factories (VF) generated in multi lamellar bodies (MLB) located in the cytoplasm. Host ER membranes surround the MLB. Newly formed virus particles decorated by spike proteins are found in a vacuole connected to the MLB. Egress of VSV-S is accompanied by the fusion of the vacuole membrane and plasma membrane.