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

Abstract

Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport 1 . 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 controversial 2-5 . Here, we quantitatively describe 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 assembly of viral-like-particles (VLPs) 6 , using fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescent microscopy (TIR-FM) in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells and typically five to six minutes 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 using conventional techniques.We monitored HeLa cells coexpressing untagged Gag and Gag fused to green fluorescent protein (GFP). This avoids the reported morphologic defect seen when Gag-GFP is expressed alone 7 and electron microscopic studies confirmed that VLPs containing Gag and Gag-GFP were morphologically indistinguishable from VLPs containing Gag only (data not shown). Gag was detected as a diffuse signal at 5-6 hours after transfection and discrete puncta that apparently localized at the plasma membrane began to appear at 6-7 after transfection ( Supplementary Fig. 1a) 4 . However, the signal from puncta at the plasma membrane was partially masked by the strong diffuse fluorescence emitted by cytoplasmic Gag-GFP ( Supplementary Fig. 1b). This diffuse fluorescence diminished relative to the intensity of the fluorescent puncta when imaged with TIR-FM, whose illumination decays exponentially from the coverslip/medium interface with a space constant of ~70 nm 8 , (Supplementary Fig. 1b). Thus, TIR-FM is well suited to monitor appearance of VLPs at the plasma membrane.Cells that had generated few (less than 20) Gag puncta at 5 to 6 after transfection were observed over the ensuing 30 to 60 min ( Supplementary Fig. 1c). Typically, 50 to 150 puncta per cell appeared during this period (movie 1). Their behavior fell into two discrete classes. One class appeared over several minutes (Fig. 1a top, b left and movie 2) and showed little lateral movement during and after emergence (average lateral velocity = 0.007 µm/sec, n=25,

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

“…Viral assembly is widely studied using HIV-Gag, the main structural protein of HIV, which is sufficient to drive the assembly of virus-like particles (VLPs) in the absence of other viral components 1,2 . Fluorescence imaging has been used to follow the time-dependent increase in the intensity of Gag clusters in living cells 3,4 , revealing the time scale of virion formation. Electron microscopy (EM) has elucidated the spatial arrangement of Gag in fully formed virions [5][6][7][8] .…”

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

“…The initial recognition of the HIV-1 gRNA by Pr55 Gag , during which discrimination against cellular and spliced vRNAs takes place, occurs in the cytoplasm and involves a very limited number of Pr55 Gag molecules [42][43][44][45] . Once these complexes become anchored in the plasma membrane, Pr55 Gag molecules are rapidly recruited as viral particle assembly proceeds 43,46 .…”

La biogenèse du VIH-1 dévoilée

“…Afin d'y remédier, nous avons travaillé avec des cellules vivantes exprimant la protéine Gag fusionnée à la protéine fluorescente verte (Gag-GFP) et utilisé une technique de microscopie qui permet d'étudier avec une très grande réso- Capturer l'assemblage des virus en temps réel : FRET et FRAP Nous avons ainsi détecté avec une réso-lution sans précédent les virus fluorescents à la membrane plasmique (Figure 1) [9]. L'une des principales difficultés était de capturer l'assemblage des virus « en direct ».…”

“…En observant des centaines de cellules à ce moment précis, et en les photographiant toutes les 5 secondes pendant 60 minutes, nous avons pu détecter l'apparition de milliers de virus fluorescents à la membrane plasmique (Figure 2A). [9]. En effet, si les particules virales sont soumises pendant leur formation à une forte application laser qui détruit irréversiblement la GFP, la fluorescence réapparaît toujours.…”

“…Une acidification rapide du cytoplasme des cellules peut être induite en exposant le milieu de culture à du CO 2 concentré à 100 % pendant 20 secondes [12]. En étudiant les conséquences de cette acidification sur la fluorescence de centaines de virus composés de milliers de copies de Gag-Phluorin, nous avons pu distinguer les virus qui étaient encore attachés à la cellule (ceux dont la fluorescence était très sensible aux changements de pH) de ceux qui avaient bourgeonné (ceux dont la fluorescence était peu sensible aux changements de pH du cytoplasme puisque séparés de la cellule) [9]. La présence des virus dans le champ après leur bourgeonnement peut s'expliquer par leur immobilisation entre les cellules et leur support ou par leur rétention à la membrane par la protéine Tetherin [13].…”