Zika virus induced cellular remodelling

Abstract

Zika virus (ZIKV) has been associated with morbidities such as Guillain-Barré, infant microcephaly, and ocular disease. The spread of this positive-sense, single-stranded RNA virus and its growing public health threat underscore gaps in our understanding of basic ZIKV virology. To advance knowledge of the virus replication cycle within mammalian cells, we use serial section 3-dimensional electron tomography to demonstrate the widespread remodelling of intracellular membranes upon infection with ZIKV. We report extensive structural rearrangements of the endoplasmic reticulum and reveal stages of the ZIKV viral replication cycle. Structures associated with RNA genome replication and virus assembly are observed integrated within the endoplasmic reticulum, and we show viruses in transit through the Golgi apparatus for viral maturation, and subsequent cellular egress. This study characterises in detail the 3-dimensional ultrastructural organisation of the ZIKV replication cycle stages. Our results show close adherence of the ZIKV replication cycle to the existing flavivirus replication paradigm.

Keywords: disease processes; infection; membrane; microbial-cell interaction; virulence; viruses (phages).

Figure 1. ZIKV growth curve and light microscopy analysis of sites of ZIKV replication during infection

A) Timecourse showing the accumulation of infectious virus particles in the supernatant from Vero E6 cells infected with ZIKV PRVABC59 at a multiplicity of infection (MOI) of 5, measured by focus-forming units (FFU). B–M) Vero E6 cells mock-infected or infected with ZIKV for 12, 24, and 48 hours (MOI=10) were fixed, permeabilized and processed for immunofluorescence using an antibody against dsRNA (J2) and DAPI. Mock-infected cells are shown in panels B–D, with virus infected cells at 12 hpi shown in E–G; at 24 hpi in H–J;, and at 48 hpi in K–M. Arrows represent examples of the punctate J2 staining observed, arcs designate likely areas of ER membrane. Nuclear labeling (DAPI) is pseudo-colored in blue, dsRNA labeling in red or white. Panels D,G,J, and M are 5X zoomed images showing J2 staining of boxed areas in panels C,F,I,L, respectively. Scale bars represent 10 μm in 1x images (scale bar in panel B), and 1 μm in 5x zoomed images (scale bar in panel D).

Figure 2. Morphology of control and ZIKV-infected Vero E6 cells

A) Representative control (mock-infected) Vero E6 cells display typical cellular morphologies in 2D EM images, including smooth and rough ER (sER, rER) Magnification bar, 250 nm. B) An image of an infected cell at 48 hpi, with a large region of convoluted membrane (CM). The CM is intercalated with actin-like filaments, and includes some spherules and virus particles that are mostly near the CM periphery (red arrowheads). There is expansion and dilation of the ER, and virus particles appear as dense, circular structures (red arrowheads mark representative viruses). Labels: virus particles (Vi; red arrowheads mark representative viruses), vesicles/spherules purported to be the site of RNA replication (Ve; blue arrows mark representative spherules), convoluted membrane (CM), ER, smooth ER (sER), rough ER studded with ribosomes (rER), multilamellar, myelin-like bodies (MLB) and mitochondria (Mito). Magnification bar corresponds to 250 nm.

Figure 3. Spherules are bounded within the ER membrane

Tomographic slices (1.1 nm thickness) highlighting ZIKV-specific sub-structures in infected cells. A) Circular spherules/vesicles appear within ER leaflets. Many spherules contain electron-dense, thread-like filaments. A′) highlights the arrangement of closely associated adjacent ER leaflets (each leaflet highlighted in yellow, orange, or brown) and spherules (blue) contained within. Dense thread-like densities are segmented in pink. B) Spherules are invaginations of the ER, with a pore opening to the cytoplasm. Shown are 1.1 nm thick sections from tomographic data, with an opening visible in B2 (segmented in B2′), but not in sections 5 nm above (B1) or below (B3).

Figure 4. Spherule, ER leaflet, and pore morphology

A) A tomographic slice (1.1 nm thickness) displaying spherules and viruses in apposed ER leaflets, segmented in A′. Spherules containing thread-like densities are segmented in blue, while those void of content are segmented in green. Virus particles (red) appear in the adjacent ER leaflet. B) 3D reconstruction showing a larger volume of this region. C) The reconstruction in B rotated 50° about the Z axis, with the virus-side ER leaflet omitted. D) Reconstruction in C rotated 90° about the Y axis, to visualize pore openings between spherules and the cytoplasm. Representative pore openings shown at arrows. White and black *’s are shown in A′–D as registration points, and in D indicate the Z plane of the images shown in A–C. Data shown are HPF/FS samples at 20 hpi. Magnification bar corresponds to 100 nm for panels A and D and 200 nm for panels B and C.

Figure 5. ZIKV envelopment, transit, maturation, and exit/entry

A) Viral envelopment intermediates observed in 1 nm thick slices from 3D reconstructions. Assembling virus particles are visible budding into the ER (A1), within the ER but still attached to its membrane (A2), and within the ER (A3); virus particles are shown at 2x (top right panels) and 3D models are shown at 0.5 x (bottom right panels). B) Virus particles (red arrowheads) accumulated within dilated regions of the ER; representative spherules marked by blue arrows. C) Inset from B shown at 2x; virus particles overlayed with red, spherules outlined with blue. D) Virus particles (Vi, and red arrowheads), observed in 10 nm thick tomographic slices, are present in the ER and Golgi. Maturation of virus depends on protein cleavage in the Golgi, suggesting these viruses are trafficking from the ER and through the Golgi. Panel B is a montage of 2D EM images. Scalebars represent 100 nm, except in A insets as noted.

Figure 6. Virus egress

A) Virus particles (red arrowhead in boxed region), apparently exiting a cell by vesicle fusion with the plasma membrane. A second virus in the neighboring cell is indicated with a red arrowhead. B) Inset from A shown at 2x. Cells are delineated by yellow and green traces of their plasma membranes in B′. Scalebar represents 250 nm in A, 125 nm in B and B′.

Figure 7. Proposed ZIKV replication pathway

As described in the text, RNA replication occurs within spherule invaginations in the ER that protect dsRNA from initiating a cellular immune response (Step 1). Viruses are assembled in dilated ER cisterna closely apposed to the site of viral RNA replication (Step 2). From the ER, viruses traffic to the Golgi, where the ZIKV prM protein is cleaved by furin, resulting in maturation of the virion (Step 3). Mature virions traffic in Golgi-derived vesicles to the plasma membrane, exiting the cell by exocytosis (Step 4).