In situ fate of Chikungunya virus replication organelles

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.

Keywords: CEMOVIS; Chikungunya; alphavirus; cryogenic-electron microscopy; electron tomography; nsP1; replication organelles; sub-tomogram averaging.

Fig 1

Characterization of CHIKV replication organelles’ sites. (A) Immunofluorescence assays of HEK293T cells infected with CHIKV for 17 h at an MOI of 50. Immunolabeling was performed using mouse monoclonal antibodies against nsP1 (anti-Alexa-488 nsp1 antibody, green channel) and dsRNA (anti-Alexa-647-dsRNA antibody, deep red channel represented in cyan). nsP3 is detected through the fluorescence of the fused mCherry protein (nsP3-mCherry, red channel). Nuclei were stained with DAPI (blue). The colocalization of nsP1, nsP3, and dsRNA reveals the presence of CHIKV replication organelles that are clearly present at the membrane of the infected cells (white arrows) and also onto cell filopodia caused by CHIKV infection (yellow arrows). Scale bar, 2.5 µm. (B–E) Thin sections of CHIKV-infected HEK293T cells (MOI of 50, 17 h post-infection) observed by transmission electron microscopy. CHIKV replication organelles appear as vesicles with diameters ranging from 50 to 100 nm, displaying condensed genetic material within its center (dark arrows), while CHIKV particles appear as smaller electron-dense dark spots (white arrows). Cytopathic vacuoles may occasionally be observed (red square and D) in which CHIKV ROs line the inner surface. A regular array is observed in the vicinity, featuring both particle-shaped structures (red arrows) and tubular structures (blue arrows).

Fig 2

CHIKV replication organelles are observed at the plasma membrane delimiting the cell body and filopodia-like extensions of infected cells. Panels A, C, and E show sections in the electron tomogram of HEK293T cells infected for 17 h at an MOI of 50, while B, D, and F display their corresponding segmentations. Scale bar, 50 nm. Plasma membrane, indicated by brown arrows, delimits the cell membrane (A) and filopodia extensions (C and E) of infected cells. CHIKV replication organelles (light brown arrows) appear as spherules of variable diameter delineated by the PM and connected to the cell by a narrow neck (white arrows). It is sometimes possible to see additional densities on their surface (yellow arrows in panels E and F). The interior of spherules contains densely packed dsRNA (light blue arrows in panels E and F). Cytoskeleton filaments present beneath the PM are indicated by purple arrows. The repetitive nature of actin filaments, present as a dense network of filaments in filopodia extensions, is clearly visible (low-pass filtered inset in panel C). Small isolated densities corresponding to cellular components are also present beneath the PM and indicated by green arrows. CHIKV particles appear as smaller electron-dense particles (red arrows) of about 50–70 nm in diameter. In the vicinity of the viral particles, local thickening of the PM can sometimes be observed (dark blue arrows in panels E and F).

Fig 3

CHIKV replication organelles diameter distribution. (A) Histogram distribution of ROs’ diameters computed from spherules located on cell bodies (red; 2,083 measurements) and filopodia-like extensions (green; 950 measurements). The inset shows how the diameter has been determined. Both populations fit with a Gaussian distribution with a mean diameter of 84.2 and 90 nm for cell body and filopodia-like extension ROs, respectively. (B) Histogram distribution of ROs’ diameters considering all ROs, corresponding to 3,033 measurements. Curve-fitting deconvolution unveils eight Gaussian sub-populations whose mean diameters are indicated.

Fig 4

Cryo-EM vitreous sections of CHIKV-infected cells reveal the presence of cytopathic vacuoles. (A and B) Some RO-filled cytopathic vacuoles (black arrows), 200–500 nm in diameter, are observed in vitreous sections. They are delineated by a lipid bilayer and often found in the vicinity of mitochondria (M). Scale bar, 50 nm.

Fig 5

CHIKV ROs display numerous patterns. Cryo-EM sections of ROs showing the base of spherules and their corresponding segmentations. ROs are displayed according to the diameter of spherules, ranging from 40 to 140 nm (from left to right) and absence (A) or presence (B) of clear associated density patterns, just beneath the PM. The PM is drawn in brown, the spherule membrane in light brown, the additional densities at the surface of some ROs membrane are in yellow, the base of ROs neck in pink, the viral RNA in blue, ribosomes in green, and intracellular filaments in purple. Viral or cell host partners present just beneath the RO neck are in green.

Fig 6

Structural organization of CHIKV RO connection to the plasma membrane revealed by sub-tomogram averaging. (A) Surface representation of the 3D reconstruction of the neck of CHIKV ROs (EMDB:EMD-50321). (B) Central section of the 3D reconstruction. As a visual aid, the location of the spherule membrane is drawn as a white dotted line. The PM appears as two parallel white linear densities encompassing a dark region. Several densities highlighted by the white and black arrows are resolved at the level of the RO neck, as well as a C-shaped density (star) corresponding to the nsP1 ring snuggly interacting with the PM at the base of the neck. (C) Central section of the surface representation of the RO connection to the PM. The dodecameric nsP1 ring atomic model (PDB: 6Z0V) fits the C-shaped ring density. The central channel is plugged by an elongated central density protruding toward the cell cytoplasm (white arrow), while a second tall barrel-like density composed of three stacked rings (black arrows) shows no apparent connection with other densities. (D) Surface representation of the map viewed from the interior of the spherule. A ring is present at the base of the spherule in which the dodecameric nsP1 atomic model fits perfectly. (E) Model of nsP1 ring insertion in the PM internal layer that supports a tight interaction of nsP1 MBO loops with the PM. The insertion of the palmitoyl moieties into the PM is indicated in red. (F) The structure of the complex composed of nsP1, 2, and 4 (PDB: 7Y38) obtained by Tan et al. (41) by single particle analysis fits well into the 3D map, revealing additional densities forming a barrel around a central density formed in part by nsP2 that could correspond to the contribution of nsP3.