Imaging the biogenesis of individual HIV-1 virions in live cells

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Abstract
For the first time, the assembly of a virus particle has been observed directly in live cells. Using techniques based on total internal reflection fluorescence microscopy, and live HeLa cells expressing fluorescent protein-tagged derivatives of Gag, the major structural component of HIV-1, individual HIV-1 virus particles were observed in real time. The resulting movies resolve a controversy over where in the cells HIV-1 assembly occurs — the virions appear individually at the plasma membrane and are assembled in about 5 or 6 minutes. This study uses several techniques based on total internal reflection fluorescence microscopy and live cells expressing fluorescent-protein-tagged derivatives of Gag, the major structural component of HIV-1, to observe and quantitatively describe the assembly of individual virus particles in real time. Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport1. However, the assembly of individual virions has never been observed in real time. Insights into this process have come primarily from biochemical analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial2,3,4,5. Here we describe quantitatively the genesis of individual virions in real time, from initiation of assembly to budding and release. We studied fluorescently tagged derivatives of Gag, the major structural component of HIV-1—which is sufficient to drive the assembly of virus-like particles6—with the use of fluorescence resonance energy transfer, fluorescence recovery after photobleaching and total-internal-reflection fluorescent microscopy in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells, and typically 5–6 min was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the determination of parameters of viral assembly that are inaccessible with conventional techniques.