Retrovirus budding is a key step in the virus replication cycle. Nonetheless, very little is known about the underlying mechanism of budding, primarily due to technical limitations preventing visualization of bud formation in real time. Methods capable of monitoring budding dynamics suffer from insufficient resolution, whereas other methods, such as electron microscopy, do not have the ability to operate under physiological conditions. Here we applied atomic force microscopy to real-time visualization of individual Moloney murine leukemia virus budding events. By using a single-particle analysis approach, we were able to observe distinct patterns in budding that otherwise remain transparent. We find that bud formation follows at least two kinetically distinct pathways. The majority of virions (74%) are produced in a slow process (>45 min), and the remaining particles (26%) assemble via a fast process (<25 min). Interestingly, repetitive budding from the same site was seen to occur in only two locations. This finding challenges the hypothesis that viral budding occurs from distinct sites and suggests that budding is not restricted laterally. In this study, we established a method to monitor the fine dynamics of the virus budding process. Using this single-particle analysis to study mutated viruses will enable us to gain additional insight into the mechanisms of viral budding.
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