Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances

Abstract

Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.

Keywords: dual-color imaging; reverse genetics; viral inclusion bodies.

Fig. 1.

NCs in the process of leaving viral inclusions do not contain VP40. (A) Huh-7 cells transiently expressing VP30-GFP were infected with MARV, fixed at 24 h p.i., and analyzed by confocal microscopy using monoclonal α-NP and guinea pig α-VP35 followed by goat α-mouse antibodies coupled to Alexa Fluor 647 and goat α-guinea pig antibodies coupled to Alexa Fluor 594. In addition, autofluorescence of VP30-GFP was recorded. A fluorescence intensity profile along the length axis of the emanating NC is displayed (diagram). (Scale bar, 2 μm.) (B) Huh-7 cells transiently expressing VP30-GFP were infected with rMARV_RFP-VP40, and fluorescence signals were analyzed by time-lapse microscopy at 29 h p.i. Time between each frame: 3 s. The time in seconds is displayed in the upper left corner of each panel. Released NC is indicated by arrows. A fluorescence intensity profile along the length axis of the emanating NC in the panel (40 s) is displayed (diagram). (C) Huh-7 cells were treated as in B but fixed at 24 h p.i., and autofluorescence was analyzed by confocal microscopy. A fluorescence intensity profile along the length axis of the emanating NC is displayed (diagram).

Fig. 2.

NCs become associated with VP40 close to the plasma membrane. Huh-7 cells transiently expressing VP30-GFP were infected with rMARV_RFP-VP40 and analyzed by confocal microscopy (A–C) or time-lapse microscopy (D). (A) NCs associated with VP40 located in filopodia are indicated by arrowheads. VP40-positive NCs, not yet recruited into filopodia (Insets), are indicated by arrows. NC without RFP-VP40 is indicated by yellow arrows. Magnified images of the boxed region (Left) are depicted in the black and white pictures. The red dotted lines indicate the cell border; fluorescence signals outside the cell border reflect filopodia-associated NCs. (B) VP40-associated NCs are more frequently detected close to the cell border than in the cell body. NCs (VP30-GFP signals, n > 400) were analyzed for their association with RFP-VP40 and distance to the cell border at 24 h p.i. **P ≤ 0.001. (C) Quantitative analysis of the ratio of VP40-associated NCs (RFP-VP40 and VP30-GFP signals colocalized) and free NCs (only VP30-GFP signal) with respect to their distance to the cell border. (D) NC in the process of becoming associated with VP40. Time-lapse microscopy of Huh-7 cells infected with rMARV_RFP-VP40 at 22 h p.i. The arrow points to the NC at the cell margins, which is magnified in the four pictures to the right.

Fig. 3.

NCs migrate with higher velocity in the cell body than in the cortex or filopodia. Huh-7 cells transiently expressing VP30-GFP were infected with rMARVwt, and the velocity of NCs was analyzed by time-lapse microscopy. NCs (n = 30) were tracked over at least four frames.

Fig. 4.

Transport of NCs is dependent on actin. (A) NCs move along actin filaments and can change to neighboring filaments. Huh-7 cells transiently expressing VP30-GFP and TagRFP-actin were infected with rMARVwt, and fluorescence signals were monitored by time-lapse microscopy. (B) Cells transiently expressing VP30-GFP and either TagRFP-actin or mCherry-tubulin were infected with rMARVwt and treated with cytoskeleton-modulating drugs. TagRFP-actin–expressing cells were incubated with 0.3 µM cytochalasin D; mCherry-tubulin–expressing cells were incubated with 15 µM nocodazole. Forty frames (one frame per 1.5 s, reflecting 60 s) of time-lapse microscopy are displayed as maximal intensity projection. Magnified pictures of the boxed regions are shown (Insets). (C) Determination of NC velocities in cells treated with cytoskeleton-modulating drugs. NCs (n = 15) were analyzed and tracked as described in Fig. 3. No directed movements of NCs were determined in cells treated with cytochalasin D.

Fig. 5.

Movement of VP40-associated NCs inside filopodia. (A) Forward and backward NC movement inside filopodia. Huh-7 cells expressing VP30-GFP were infected with rMARV_RFP-VP40 and analyzed at 29 h p.i. by time-lapse microscopy. Forward movement is indicated by arrows; backward movement is indicated by arrowheads. Cell border is indicated by dotted lines. Only VP40-associated NCs (yellow) were detected in filopodia and moved bidirectionally (velocity 100 nm/s). Backward movement requires a turn of the NCs (between 2 and 5 s). (Lower Left, Inset) (5 s) Maximal intensity projection reflects 6 s. (B) Cotransport of Myo10 with NCs inside filopodia. Huh-7 cells transiently expressing GFP-Myo10 were infected with rMARV_RFP-VP40. NCs at the tip of a filopodium are indicated by arrows. Colocalization of NCs with GFP-Myo10 is indicated by arrowheads.