There is abundant evidence in the literature that a perturbation of normal cellular levels of the vacuolar protein sorting (VPS) machinery results in defective endosomal sorting of receptors (3
). Likewise, a perturbation of the levels of TSG101 and other VPS components clearly results in inefficient HIV-1 particle release (9
). What happens when both of these processes need to take place simultaneously in the same cell? Do both processes utilize and/or compete for the same cellular pool of VPS machinery? If so, is any one of the two processes compromised in efficiency? In this study, we addressed these questions directly by monitoring ligand-induced downregulation of EGFR in cells expressing HIV-1 Gag alone or along with other viral proteins. We demonstrated that the rate of receptor downregulation is decreased when HIV-1 Gag is present in the cell.
COS-1 cells were used in this study as a model system for the demonstration of proof of principle. Although COS-1 cells are not authentic targets of HIV-1 infection, they exhibit three important features that were relevant for this study. First, these cells are permissive for HIV-1 assembly and particle production. Second, COS-1 cells express abundant levels of endogenous EGFR. Third, the envelope-mediated cell death and cell fusion that are induced by HIV-1 infection of target cells do not occur in Gag-expressing COS-1 cells. We were therefore able to demonstrate a correlation between the amount of HIV-1 Gag expressed and the amount of endogenous EGFR remaining after EGF stimulation on a single-cell level. As depicted in Fig. , there was a positive correlation between Gag and EGFR fluorescence intensities over a seven- to eightfold range of Gag expression levels.
Although it is now known that approximately 5,000 Gag molecules are packaged per immature HIV-1 virion (4
), the amount of cellular Gag that is expressed on a per-cell basis in an HIV-1-infected cell has not been reported. It is likely that Gag expression levels vary considerably during the course of the infection. However, two lines of evidence suggest that our findings with Gag-transfected COS-1 cells may be applicable to the situation that occurs in HIV-1-infected cells. First, equivalent amounts of Gag protein were produced when expression was driven from a heterologous promoter (CMV) or from the HIV-1 LTR (pHXB2ΔBalD25S) (Fig. ). Second, the pattern of pEGFR staining in cells transfected with pHXB2ΔBalD25S was similar to that observed for cells transfected with pCMV5 Gag-GFP (Fig. ). Thus, we hypothesize that it is possible for the HIV-1 LTR to produce the levels of Gag expression that are needed to attenuate EGFR downregulation.
The scenario during an actual HIV-1 infection, however, may be much more complicated. The presence of other viral proteins may serve to either further enhance or mask Gag-mediated effects on receptor trafficking and signaling (23
). It would therefore be interesting to further investigate whether the ESCRT-dependent endosomal sorting of receptors is perturbed in HIV-1-infected target cells, e.g., T cells. A potential candidate receptor in the T cell is CXCR4, which has been shown to be dependent on ESCRT components for downregulation (18
Interestingly, the effect of HIV-1 Gag overexpression on receptor downregulation was not as strong as one would expect if the virus completely usurped the cellular machinery and redirected it towards sites of viral egress. This is emphasized by the lack of clustering of endosomal compartments that are normally observed when the cellular levels of VPS components are perturbed. There are three potential explanations for this observation. (i) HIV-1 Gag induces an increased expression of ESCRT proteins to accommodate its needs. This possibility is unlikely, since no differences in endogenous TSG101 protein levels were observed for HIV-1 Gag-transfected versus untransfected cells (not shown). (ii) The cellular TSG101/ESCRT pool is too large to be completely depleted by the overexpression of Gag/PTAP-containing proteins. (iii) The stoichiometry of the Gag-ESCRT interaction is not 1:1. The number of ESCRT complexes that are recruited per Gag assembly unit in vivo is not known. The multimerization of Gag into assembly complexes, which consist of ~5,000 Gag molecules, could physically restrict its ability to sequester large, 350-kDa ESCRT complexes.
An obvious consequence of altered receptor trafficking is altered intracellular signaling. As depicted in Fig. , cells expressing HIV-1 Gag exhibited increased EGFR-mediated signaling, as evidenced by hyperactivation of the ERK/MAP kinase pathway. This finding is consistent with the presence of increased amounts of pEGFR in endosomal compartments 180 min after the addition of EGF. The ability of endosomal EGFR to induce intracellular signaling has been documented (31
). Changes in intracellular signaling are a well-established characteristic of HIV-1 pathogenesis. The binding of HIV-1 gp120 to CD4 and a chemokine receptor (CXCR4 or CCR5) during virus entry triggers a plethora of signaling pathways (21
). The upregulation of cellular signaling regulates the ability of the virus to replicate efficiently within the target cell (21
). After provirus integration into the host genome, either a productive or a latent infection can be established. The switch from a latent to productive infection has been shown to be triggered and maintained by extracellular mitogen/agonist-induced MAP kinase activation through the Ras/Raf/Mek signaling pathway (32
). MAP kinase stimulates AP-1, which, along with NF-κB, transactivates the HIV-1 LTR (32
). The activation of MAP kinase also increases the infectivity of the HIV-1 virions that are subsequently produced (33
). The observations presented in this study strongly suggest that Gag-mediated budding and egress during the late stages of the viral life cycle may indirectly contribute to enhanced HIV-1 replication and/or infectivity by slowing down mitogen-induced cell surface receptor downregulation.