The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex

Abstract

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV(KUN)) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV(KUN) replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV(KUN) replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.

FIG. 1.

The WNV_KUN RC contains only high-mannose conjugated glycans. IF analysis of Vero cells mock infected (a to f) or WNV_KUN infected (g to l), immunolabeled with antibodies to dsRNA, and costained with Alexa Fluor 488-conjugated ConA or Alexa Fluor 594-conjugated WGA is shown. Colocalization is observed only in i as a yellow hue.

FIG. 2.

Both host and viral glycoproteins residing within the WNV_KUN RC appear to be sequestered at a pre-Golgi step. Costaining with Alexa Fluor 488-conjugated ConA (b and h) or Alexa Fluor 594-conjugated WGA (d and j) and either the host glycoprotein GalT (a and e) or the viral glycoprotein NS1 (g and k) reveal that both marker proteins are dually labeled only with Alexa Fluor 488-conjugated ConA and not Alexa Fluor 594-conjugated WGA. These results suggest that the RC-resident glycoproteins have not transited through the Golgi apparatus, where further processing and the addition of complex glycans would have occurred.

FIG. 3.

The WNV_KUN RC is intimately associated with the RER membranes and is open to the cytoplasm via a membranous neck. (A) At 48 h posttransfection, KUNrepMxA-transfected tetKUN-CprME cells were fixed for resin embedding and analyzed by electron tomography. A VP utilized for the subsequent 3D surface model is shown. (B) Visualization of the vertical stacks revealed the presence of “neck-like” structures (indicated by arrows) that tether the individual vesicles to the membrane. (C) 3D surface model of the WNV_KUN RC reveals the intimate association of the individual vesicles housing the viral RNA (indicated in yellow) with the RER membrane (indicated in red) that is decorated with associated ribosomes (indicated in white and additionally highlighted with asterisks in A and B). (D to H) Rotational views of the WNV_KUN RC highlighting the pores connecting the vesicles to the cytoplasm and the spatial arrangement of the vesicles within the VP. The 3D model was constructed by using median filtering and automatic threshold segmentation using IMOD software.

FIG. 4.

Vesicles within the WNV_KUN RC are connected to each other directly via pores. (A to C) 3D surface modeling revealed that vesicles within the internal space of the VP, not directly in connection with the bounding membrane, are fused to neighboring vesicles via pores (highlighted by arrows in A). The individual vesicles are indicated in white, and the ER membrane is depicted in red. C is a 90° rotation (or top view) of the VP in B. (D and E) Snapshots collected from the tomogram shown to highlight the “necks” or “pores” that link individual vesicles within the RC. Connections are indicated by arrows.

FIG. 5.

Structural modeling of the viral RNA within the vesicles reveals a complex structure closely aligned to the vesicle pore. Two individual vesicles were 3D surface modeled (A, C, and E and B, D, F, and G, respectively), and the visible RNA was also surface rendered. The analyses showed that the viral RNA is present in a dynamic state and may reveal the presence of individual tertiary stem-loop structures present on the RNA backbone. Our analyses have also revealed that the viral RNA is juxtaposed to the pore-like opening tethering the individual vesicles.

FIG. 6.

The majority of viral RNA within the VP is dsRNA. (A and B) Representative images of WNV_KUN-infected Vero cells without (A) or with (B) RNase A treatment under high-salt conditions. As can be observed, the majority of vesicles within both samples contain visible threads. (C and D) Representative images of WNV_KUN-infected Vero cells without (C) or with (D) RNase A treatment under low-salt conditions. As can be observed, the majority of vesicles within C contain visible threads, which is in stark contrast to the vesicles in D, where very few threads are observed. In all cases, the magnification bars represent 200 nm. (E) Quantitative analysis of the percentage of vesicles containing visible threads under all conditions. The results were calculated from duplicate experiments, and the statistical analysis was performed by using an unpaired t test with a 95% confidence interval.