ESCRT machinery components are required for Orthobunyavirus particle production in Golgi compartments

<div><p><i>Peribunyaviridae</i> is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus <i>Orthobunyavirus</i> that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.</p></div

OROV viral factories derive from Golgi membranes.

<p>(A) Mock infected and (B) OROV infected HeLa cells at 18 h p.i.. White arrowheads show dilated Golgi cisternae. (C) Boxed area in (B) showing virus inside the Golgi cisternae (black arrowheads). 3D-SIM of mock infected (D) and OROV-infected (E) HeLa cells imaged at 18 h p.i. Cells were stained to OROV, TGN46 and giantin proteins, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007047#ppat.1007047.g002" target="_blank">Fig 2</a>. The images represent projections of Z stacks (125 μm each) of cells after deconvolution. (F) Inset representing the boxed area of E. (G) Immunoelectron micrograph using a polyclonal anti-OROV antibody of an OROV infected cell at 24 h p.i. (g’-g”) Boxed areas in (G) showing virus staining on the vesicle membrane (white arrow) and viral particles inside the vesicle (black arrow). GC = Golgi complex; m = mitochondria; pm: plasma membrane; ER: Endoplasmic reticulum.</p

Vps4A is recruited to the TGN46 positive structures during OROV assembly.

<p>HeLa cells were infected with OROV and transfected with Vps4Awt-GFP (shown in red, A-J) or Vps4AE/Q-GFP (shown in red, K-O) plasmids. After 24 h p.i, cells were fixed, double stained with anti-OROV (shown in green to facilitate comparison with other Figures) and anti-TGN46 (Cyan) antibodies. (A-D) HeLa cells expressing Vps4wt-GFP were fixed and analyzed by conventional confocal microscopy. (F-I) 3D-SIM images of HeLa cells processed as (A-D). The image represents a projection of Z stacks (125 μm each) of cells after deconvolution. (K-N) HeLa VpsE/Q-GFP expressing cells were fixed after 24 h p.i and analyzed by conventional confocal microscopy. Bars = 10 μm. (E, J and O) Insets representing the boxed areas of A-D, F-I and K-N, respectively. Bars = 2 μm. (P) The areas of Vfs from least 15 cells for each condition from three independent experiments were determined using ImageJ software and are shown as mean ± SEM ***, P < 0.0005 (two-tailed paired <i>t-</i>test).</p

Alix is recruited to TGN during OROV assembly.

<p>Mock infected (A-D) or OROV infected (F-I) HeLa cells were transfected with VCt-Alix and VNt-Alix plasmids (red). Cells were immunostained to OROV proteins (green) and TGN46 (cyan) and analyzed by confocal microscopy. (K-N) 3D-SIM images of HeLa cells processed as (F-I). The image represents a projection of Z stacks (125 μm each) of cells after deconvolution. Bars = 10 μm. (E, J and O) Insets representing the boxed areas of A-D, F-I and K-N, respectively. Bars = 2 μm.</p

OROV recruits Golgi complex and TGN proteins to its assembly site.

<p>HeLa cells were infected with OROV and fixed after 0 h (A-D) or 24 h p.i. (F-I). Golgi complex and TGN proteins (Giantin and TGN46, respectively) were co-immunostained with OROV proteins and analyzed by confocal microscopy. Bars = 10 μm. (E and J) Insets representing the boxed areas of A-D and F-I, respectively. Bars = 2 μm.</p

Knockdown of Tsg101 and Alix disrupt OROV assembly.

<p>HeLa cells were transfected with control, Tsg101#1 or Alix#1 siRNAs and infected with OROV (MOI = 1). (A-C) After 24 h p.i, cells were stained with anti-OROV antibody and analyzed by confocal microscopy. Bars = 10 μm. (D, E and F) Zoomed images of the boxed areas shown in A, B and C, respectively. White arrows indicate OROV Vfs. Bars = 2 μm. (G) The areas of Vfs from least 15 cells for each condition from three independent experiments were determined using ImageJ software (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007047#sec009" target="_blank">Materials and Methods</a>) and are shown as mean ± SEM. (H-J) After 12 p.i., cells were collected and processed for immuno-electron microscopy staining with anti-OROV antibody. (K, L) Area of the viral vesicles (K) and number of viral-like particles within viral vesicles (L) were obtained using ImageJ software (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007047#sec009" target="_blank">Material and Methods</a>). Bars represent the mean and ± SEM from a total of 13, 19 and 17 vesicles for siControl, siTsg101 and siAlix samples, respectively, in least 10 different cells for each condition. **, P < 0.005; ***, P < 0.0005. (two-tailed paired <i>t-</i>test).</p

OROV replication cycle.

<p>HeLa cells infected with OROV were analyzed during the virus cycle. (A) Virus titers were determined by TCID₅₀ assay in Vero cells. Data are the mean ± SD of three independent experiments and are displayed as TCID₅₀/mL. (B) Detection of OROV proteins in cell lysate and supernatant at different times p.i. by immunoblotting. Primary antibodies to OROV proteins and GAPDH (loading control) were used and the molecular weight (in kDa) is indicated on the left. A representative immunoblot from three independent experiments is shown. (C-G) Subcellular distribution of OROV proteins (green), monitored by immunofluorescence and confocal microscopy analysis. The images shown are representative of the period p.i. indicated. Cell outlines are indicated by dashed lines. Nuclei were stained with DAPI (blue). Bars = 10 μm.</p