We have applied tetracysteine tagging and biarsenical labeling to examine the location of Gag in live cells. Using this approach, we found that steady-state Gag, expressed either alone or within the context of a pol− virus, was present mostly at the plasma membrane with little protein found associated with intracellular compartments. The FlAsH-binding tag had no observed effect on localization or VLP production of Gag: both Gag and Gag-TC were distributed in a similar pattern in our model HeLa cells as observed by FlAsH labeling and antiserum staining. Moreover, electron microscopy in 293T cells revealed virus assembly taking place at the plasma membrane with assembly structures only rarely found in the cytoplasm, consistent with the findings from our fluorescence imaging. Thus, Gag-TC faithfully recapitulates the properties of Gag and reveals that Gag localizes mostly to the plasma membrane at steady state.
Using a fluorescent two-color technique, we found that newly synthesized Gag also accumulates primarily at the plasma membrane within 30 min of synthesis. Thus, it is likely that the majority of Gag is primarily synthesized at or very near the inner leaflet of the plasma membrane. This corresponds well with a previous biochemical report that assembly-competent Gag associates with membranes within 10 min of synthesis (69
). Both of these results, the rapid membrane binding and our plasma membrane localization of Gag, are consistent with our previous proposal that Gag translation, RNA packaging, and assembly are spatial and temporally linked at or near the plasma membrane (54
Two-color analysis also showed that both existing and nascent Gag proteins are mostly found in the same limited regions of the plasma membrane (Fig. , all merge), supporting the concept that HIV-1 egress occurs through defined regions of the plasma membrane, possibly through late endosomal membranes (19
) and/or cholesterol-rich regions such as lipid rafts (34
). This may also be due to the presence of the appropriate endosomal sorting machinery, e.g., ESCRT complexes, at these sites (15
One point to consider in interpreting any Gag localization study is that not all Gag proteins produce particles: a sizable fraction of synthesized Gag (25 to 80%) fails to form into particles and is degraded (43
). One fate of defective Gag is degradation by the proteasome pathway (63
). Thus, it is unlikely that all of the Gag molecules observed by any fluorescence imaging technique are functional assembling molecules. Despite this caveat, the predominant localization of Gag to the plasma membrane observed by our fluorescence and electron microscopy makes it likely that some of the Gag proteins we observe by fluorescence were in the process of assembly and budding.
Rous sarcoma virus Gag can pass through the nucleus and might select its genomic RNA for packaging during this journey (62
). Since the matrix region of HIV-1 Gag has a nuclear localization and export signal (10
), it is possible that HIV-1 Gag could act similarly. However, we did not observe any specific nuclear staining in any of our experiments. Therefore, any nuclear import or export would involve only a small percentage of HIV-1 Gag that is beyond our limit of detection.
Our FlAsH-labeled imaging of Gag expressed from a full-length HIV-1 pol−
molecular clone found that Gag assembling into virions was distributed similar to Gag-TC and Gag. Thus, the expression of other viral proteins that are involved in assembly and have been previously implicated in Gag localization, primarily Env (36
) and Vpu (22
), the presence of cis
-acting RNA sequences (68
), or levels of Gag expression did not appreciably alter the Gag localization pattern in HeLa cells.
Unlike the other tagged constructs, the Gag-LTC construct localized more to internal structures than at the plasma membrane. Staining for CD63 and Lamp2 markers demonstrated that these Gag-containing structures were late endosomes or MVBs. This alternate localization pattern did not alter the release of Gag-LTC versus Gag-TC or cause any observable increase in internal particle assembly by electron microscopic analysis. Perhaps these late-endosomal-associated Gag molecules are degraded as discussed above. While the residues flanking the tetracysteine motif (underlined) (GSDSSGSMHVVDSSGSMPCCPGCCGS) in Gag-LTC do not have any obvious intracellular targeting sequences, they appear to be responsible for its increased intracellular accumulation when placed at the C terminus of Gag. This finding suggests that caution should be used when producing Gag fusion proteins for imaging; it is important to validate the localization of Gag or any protein that contains even minimal additional sequences.
We applied biarsenical labeling to study the localization of two different assembly mutants. Blocking Gag myristylation caused Gag-LTC to lose plasma membrane and late endosomal targeting and exhibit a generalized cytoplasmic localization. These data essentially agree with those previously obtained by immunofluorescence of HIV-1 Gag myristylation mutants in African green monkey (66
), Cos-7 monkey (25
), and HeLa cells (49
), as well as the imaging of Gag-GFP in the presence of a myristylation inhibitor (25
). Therefore, whatever the role for myristylation in Gag localization, it is essential for plasma membrane and late-endosomal targeting, likely due to an early event in Gag localization.
The mutation of the PTAP sequence in Gag-LTC did not apparently alter its localization compared to the wild type: both plasma membrane and interior vesicular sites contained Gag with little difference in CD63 overlap. This is consistent with recent findings by Ono and Freed, who found little difference in localization between wild-type Gag and a p6Gag
deletion mutant (47
). Similar results have also been obtained for an Ebola VP40 L-domain mutant (32
). Together, these results suggest that the defect in L-domain-mediated budding in HeLa cells occurs mostly at the plasma membrane rather than due to a deficiency in Gag trafficking. Thus, Tsg101 and other L-domain-interacting proteins more likely act in the budding process at the plasma membrane immediately during release, as previously proposed (2
), rather than acting in a sorting process into the late endosome/MVB pathway (19
While fusing proteins of interest with fluorescent proteins (FPs) from various Cnidarians is the workhorse for imaging proteins (70
), the addition of a short tetracysteine tag and biarsenical labeling is emerging as an alternate approach to observing proteins in living cells. An important advantage to this method is that the tetracysteine hairpin can bind biarsenical dyes rapidly after synthesis (30
), so newly synthesized Gag can be visualized. In contrast, FPs need to fold and autooxidize to form a functional chromophore after synthesis, processes with combined experimental half-life values ranging from 27 min to 4 h (23
). Given the rapid kinetics of nascent Gag for membrane binding (starting at 10 min) and budding (starting at 1 h) (64
), the tetracysteine tag allowed us to visualize the earlier stages of Gag assembly.
Despite these advantages, we have observed intermittent background problems, mostly internal staining that remains a significant problem. However, with the proper controls, it is manageable. Thus, while the biarsenical labeling is clearly a valuable tool, it remains more technically demanding than FP techniques. Current research aimed at optimizing the sequences flanking the tetracysteine motif promises to bring even greater sensitivity and lower backgrounds. Additionally, while the primary motif itself did not alter the properties of Gag, in Gag-LTC, some of the sequences that flank the tetracysteine tag did affect the localization of Gag. Therefore, tagged and untagged proteins need to be compared in some fashion to rule out this potential problem.
One of the important challenges for retrovirology is to understand the spatial organization of the viral proteins within the cell and the cellular components that interact with the various viral proteins during assembly and budding. We are currently introducing biarsenical tags into various proteins in replication-competent HIV-1 to study their localization in live primary cells. The approach that we have developed here should assist this effort and promises to allow us to study HIV-1 Gag, Pol, and Env translation and trafficking in both model and primary cell systems. In turn, this should assist our understanding of HIV-1 assembly and biology.