Monitoring RNA release from human rhinovirus by dynamic force microscopy

Abstract

Human rhinoviruses were imaged under physiological conditions by dynamic force microscopy. Topographical images revealed various polygonal areas on the surfaces of the 30-nm viral particles. RNA release was initiated by exposure to a low-pH buffer. The lengths of the RNAs that were released but still connected to the virus capsid varied between 40 and 330 nm, whereas RNA molecules that were completely released from the virus were observed with lengths up to 1 micro m. Fork-like structure elements with 30-nm extensions were sometimes resolved at one end of the RNA molecules. They possibly correspond to the characteristic multi-stem-loop conformation, the internal ribosomal entry site, located at the 5' region of the genome. This study demonstrates that dynamic force microscopy can be used to study viral RNA release in situ under physiological conditions.

FIG. 1.

Topography images of HRV2 immobilized on mica. (A) Densely packed HRV2 monolayer. Dark regions correspond to holes, and white spots are virus particles bound on top of the first layer. The image scan size was 6 μm. (B) Cross-sectional profile of a homogeneous virus monolayer containing a large defective hole. Using uncovered mica as a reference, we determined the height of the monolayer to be 31 ± 3 nm. (C) Small scan size image of a densely packed HRV2 monolayer. The close apposition of the virions results in some structural deformation of the capsids. On most of the capsids, a regular pattern of ∼3 nm can be observed. The image scan size was 100 nm. (D) Separated virions show polygonal areas without deformation of the capsid. The image scan size was 250 nm.

FIG. 2.

RNA release at low pH. (A) Single virions adsorbed onto mica and imaged in a pH 7.6 buffer. The scan size was 900 nm. (B) The pH of the buffer was changed to 4.1 and maintained for 2 h. Images were acquired again at pH 7.6. Virus particles are observed together with RNA molecules (arrows), either separated from the virus capsid or still connected. The scan size was 900 nm.

FIG. 3.

Partially released RNA molecules. (A) Bent RNA molecules in connection with the virus capsid. Virus particles are observed as bright spots with heights of about 31 nm. The smaller bright dots with 10-nm diameters are presumably debris of the virus capsid. The scan size was 200 nm. (B) Regularly shaped virus particles with straight RNA molecules still connected to the virus capsids. The scan size was 250 nm. (C) Single virus particle with an attached, straight RNA molecule observed at a scan size of 100 nm. (D) Bent RNA molecule protruding from the virus capsid observed at a 150-nm scan size. (E) Distribution of the contour lengths of RNA molecules connected to the virus capsids. In total, the contour lengths of 24 RNA molecules were used to construct the histogram.

FIG. 4.

Enzymatic digestion of released RNA. (A) RNA molecule in the process of extrusion (arrow) after incubation at pH 4.1. The scan size was 300 nm. (B) Same scan area as for panel A after gentle injection of RNase A into the liquid cell and incubation for 10 min. (C) Completely (upper right arrow) and partially (lower left arrow) released RNA molecules appear after incubation at pH 4.1. The scan size was 900 nm. (D) After injection of RNase A into the liquid cell, the RNA molecules disappeared. (E) Time series of images showing completely and partially released RNA molecules. The images were acquired in the absence of RNase and demonstrate that the imaging process in no way displaced or modified the fibers (arrows).

FIG. 5.

Fully released RNA molecules. (A) RNA molecules completely released from virus capsids were occasionally obtained with a fork-like structure at one end of the RNA (box). The length of the fork was ∼30 nm. The scan size was 200 nm. Note that the RNA molecule is not in the process of extrusion but only touches the virus capsid from the side (arrow). (B) Individual RNA molecule separated from the virus capsid and exhibiting a similar fork-like structure (box) to that in panel A. The scan size was 150 nm. (C) Tightly packed RNA molecules on mica obtained upon exposure of the virus to a low pH at a high surface coverage. Several virus particles can be seen as bright white spots, and RNA molecules are tightly packed, forming a layer with a height of 1.5 nm. Isolated RNA molecules were observed only occasionally, exhibiting lengths of ∼1 μm (arrow). The scan size was 2 μm.