Fluorescent protein fusions provide a powerful methodology to reveal dynamic intracellular protein-trafficking pathways. However, a prerequisite to interpreting the significance of GFP fusion experiments is to determine the protein's ability to function in place of the native protein. To follow the dynamics of HIV Gag during assembly and transmission, we present the first autonomously infectious, provirally expressed Gag-GFP fusion. An iGFP insertion strategy minimizes disturbances to viral fitness. This tool allowed us to track the distribution and oligomerization of Gag during the formation of infectious virus particles without helper virus. The remarkable infectivity of the virus validates its use for studying Gag dynamics.
Immunofluorescence with anti-p17 and anti-p24 antibodies revealed that the localization of HIV Gag-iGFP was nearly identical to that of native HIV-1 Gag. Interestingly, however, the fluorescence at the membrane from GFP was much more pronounced than that seen by immunofluorescence. A recent study has demonstrated that many antibodies do not readily react with oligomerized forms of Gag that may be concentrated at the plasma membrane (32
). It therefore is possible that standard immunostaining approaches routinely underestimate the levels of Gag at the plasma membrane. HIV Gag-iGFP will be useful in developing and testing quantitative models for Gag transport and viral assembly. Our FRET studies, discussed further below, confirmed that comparatively low levels of antibody reactivity at the plasma membrane correlated with intense Gag oligomerization at that site. As an additional consideration, the GFP tagging also may preferentially reveal more mature, or well folded, pools of Gag that are concentrated at the plasma membrane. The results further establish fluorescence tagging as an important adjunct to immunofluorescence for determining the cellular distribution of Gag.
The high level of infectivity is achieved in part by introducing GFP into the Gag precursor protein flanked by HIV protease cleavage sites, which effectively are processed in mature virus particles. The resulting processed virus particle thus carries unperturbed MA and CA, which limits the negative effects on viral infectivity. Single-round infectivity experiments showed no defect in infectivity of the HIV Gag-iGFP virus. We found that in a highly permissive T-cell line, MT4, the virus actually could replicate rapidly with full cytopathic effects. The spread of the virus in culture was further demonstrated by the requirement for ongoing reverse transcriptase activity to fully infect the culture. In other T cells, such as Jurkat cells and peripheral blood mononuclear cells, infection was observed at similar initial titers, but we did not observe rapid spread. These results suggest that the spacing between MA and CA is important for propagation in culture for certain T-cell lines.
This fusion strategy also allowed us to assess the extent to which the localization of the Gag precursor in producer cells is altered by the activity of HIV protease. Although cleaved Gag-iGFP was found in all cell lysates, intracellular protease activity did not alter the localization of Gag-iGFP in most cells. From this, we infer that protease is active only within virus particles, where cleaved GFP cannot diffuse. Our live imaging further suggested that intracellular cleavage of diffusible Gag occurs in the latest stage of viral gene expression in a small percentage of cells. We suggest that in these cells a diffuse GFP fluorescence and rounded morphology are indicative of the terminal effects of intracellular HIV protease activation (50
). We conclude that HIV Gag-iGFP permits tracking of full-length unprocessed Gag within the majority of virus-producing cells, without complications of premature intracellular processing of Gag-iGFP.
Our FRET assays show a strong homooligomerization of Gag at the plasma membrane. FRET signal also was detected at intracellular vesicular compartments. Two previous FRET studies that examined Gag-GFP oligomerization in the absence of other viral proteins (7
) also found that Gag was strongly oligomeric at membrane sites. These studies differed in their view of whether any lower-order oligomerization occurs in the cytoplasm. With an NFRET index and acceptor photobleaching analyses, we show that nonvesicular Gag-iGFP within the cytoplasm is engaged in FRET. These studies also reveal that intense membrane FRET is dependent upon key residues within the I domain. Previous biochemical studies have speculated that prior to membrane engagement, Gag forms low-order oligomers (21
). Weak cytoplasmic FRET may be indicative of these assembly intermediates. A previous study of Gag-GFP did not observe nonvesicular, cytoplasmic FRET (7
). Our contrasting result may be explained by the presence of other viral genes or may be due to differences in the construct design. Inserting GFP internally into Gag may create an organized lattice of GFP constrained between MA and CA that may enhance its ability to induce FRET. The high sensitivity of the Gag-iGFP FRET assay makes it attractive for use in high-throughput assays to identify inhibitors of HIV assembly.
GFP has revolutionized cell biological studies by enabling researchers to dynamically track the movements of proteins in living cells. This study validates a novel GFP fusion approach to track HIV Gag during the course of viral infection. Our single-cell, four-dimensional time-lapse fluorescence microscopy studies showed the progression of Gag distribution in cells through four discreet stages (Fig. ). The progression went from a diffuse cytoplasmic localization, to a pattern with additional plasma membrane accumulation, to later stages in which intense plasma membrane staining coincides with accumulations at intracellular vesicular compartments. Finally, in the most intensely expressing cells, we see nuclear invasion, loss of discreet compartments, and rounded cell morphology. Because we observe different patterns at different stages, we speculate that the intracellular dynamics are altered in a switch-like manner depending on the concentration of Gag (37
) or other viral proteins.
There has been considerable debate regarding the pathway of Gag trafficking to the plasma membrane. Many suggest that the late endosome is a requisite trafficking step prior to Gag engagement of the plasma membrane (8
). Recently, others have presented compelling data to suggest that Gag directly engages the plasma membrane (17
). We show with continuous single-cell live imaging that as levels of Gag increase in a cell expressing all viral genes from a natural proviral context, accumulations of Gag at the plasma membrane precede accumulations of Gag at intracellular vesicular sites. As suggested recently, intracellular vesicle-associated Gag may represent endocytosed protein from the cell surface (11
). Our live-imaging results with HIV Gag-iGFP are supportive of the view that localization of Gag to the plasma membrane does not require a late endosomal intermediate (17
). The acquisition of full three-dimensional data sets at each time point also allowed us to resolve that these intracellular accumulations of Gag were not contiguous with the plasma membrane, as has recently been described for macrophages (6
). Further analysis of HIV Gag-iGFP in other cell types may help to determine the extent to which Gag targeting and assembly are controlled differently according to cell type.
Lastly, we find that not only are the infected cells fluorescently labeled but the progeny viruses are also stoichiometrically endowed with green fluorescence. This virus will be particularly useful for visualizing intercellular structures that facilitate cell-to-cell transmission, called virological synapses (16
). An accompanying paper in this issue illustrates the utility of HIV Gag-iGFP in quantifying the efficiency of cell-to-cell transfer between HIV-infected and uninfected T cells (4
). Although the virus is not replication competent in all cell types, HIV Gag-iGFP still may provide a sensitive method to track the movements of infected cells and viral particles in animal models. Ultimately, a clearer understanding of the events that link assembly with viral transmission will be obtained by directly observing the events with live imaging of infected immune cells.