While many cells do not constitutively express tetherin, its expression can be induced by IFN-α (5
), and as such, tetherin is likely part of a general IFN-induced antiviral response. Many viral infections could potentially trigger tetherin expression, which could in turn inhibit virus dissemination. Here, we show that tetherin is capable of inhibiting the release of a variety of VLPs assembled using the structural proteins of retroviruses and filoviruses. While we have obviously not surveyed all enveloped viruses, the viral proteins used herein included those that have no sequence homology to one another, are from different virus families, are targeted to cell membranes in different ways, and exhibit different morphogenesis pathways. The fact that the release of all VLPs examined was inhibited, at least to some extent, suggests that tetherin might be a very broad antiviral factor that restricts the spread of many envelope viruses. In particular, these findings suggest that tetherin functions by targeting a component of retroviruses and filoviruses that is shared among them even in the absence of viral protein sequence homology. This idea, coupled with the fact that the tetherin protein essentially consists of two membrane anchors linked by a predicted coiled coil, makes attractive the notion that tetherin restricts particle release by “cross-linking” the viral and host lipid bilayers after budding. The striking colocalization of tetherin with VLPs at the plasma membrane is also consistent with such an idea.
The inhibition of virion release by tetherin did not lead to a dramatic accumulation of cell-associated viral proteins. The reasons for this are not clear but suggest that the VLPs that are retained by tetherin are destroyed at rate that exceeds or is not greatly different from that of their synthesis. This would likely be through endocytosis, which we and others have previously shown to be the fate of Vpu-defective HIV-1 (12
), followed by lysosomal degradation. Moreover, it may simply be the case that only a fraction of the viral protein that is synthesized is actually released as particles. Thus, the amount of viral protein that is observed in cell lysates would be determined by its intrinsic turnover rate rather than particle release versus retention.
Many enveloped viruses that bud through the plasma membrane might have been placed under evolutionary pressure to avoid or antagonize tetherin activity, as does HIV-1 by expressing Vpu. In this study, we deliberately generated VLPs using minimal viral components wherever possible to avoid the potentially confounding variable of undiscovered antagonists that might obscure tetherin activity. Nonetheless, our findings beg the question of how viruses that are intrinsically sensitive to tetherin might avoid its activity. There is at least one other example of a potential tetherin antagonist, namely, the K5 protein of Kaposi's sarcoma-associated herpesvirus, which is able to reduce steady-state levels of tetherin when overexpressed (2
). Thus, it is conceivable that accessory proteins of unknown function that are encoded by several complex retroviruses could include those that exhibit antitetherin activity. Additionally, some reports suggest that certain Env proteins, particularly those from HIV-2 and certain HIV-1 strains, exhibit Vpu-like activity (6
) and could, therefore, represent tetherin antagonists. Since Env proteins are, by definition, membrane proteins, one could envisage that other viral Env proteins could possess such activities. Among the VLPs tested, MPMV was the only one for which a full-length viral genome was used to generate VLPs because previous reports indicated that Env is required for the transport of capsids to the plasma membrane for envelopment (31
). Curiously, particles generated by this construct appeared to be somewhat less sensitive to low levels of tetherin than those generated by Gag proteins of other retroviruses. Whether this is an intrinsic property of MPMV particles or is due to the presence of a tetherin antagonist in MPMV (e.g., Env) is unknown. However, we did notice that levels of tetherin expression appeared to be reduced in cells transfected with a mixture of MPMV proviral and Gag-GFP expression plasmids, hinting at the latter possibility.
Tetherin could also be avoided by viruses in the absence of a direct antagonism of its function. Viruses that do not induce a strong IFN response or attenuate the IFN response through the action of viral inhibitors may not require a tetherin antagonist, particularly if they replicate in cells or tissues that do not ordinarily express tetherin. Another possible way for enveloped viruses to avoid tetherin would be to bud through membranes, or membrane domains, from which tetherin is absent. Tetherin harbors a glycosylphosphatidylinositol anchor and may therefore be concentrated into putative cholesterol-rich domains at the plasma membrane (15
). Several enveloped viruses, including Eb and HIV-1, have been reported to assemble at cholesterol-rich plasma membrane domains (3
), and colocalization analysis of most nascent retrovirus and filovirus VLPs with tetherin revealed a good correlation between the presence of viral protein and tetherin signals at the plasma membrane. Thus, if it is indeed the case that particular membrane microdomains are selected by viruses for budding, then tetherin's localization might facilitate encounters with nascent virions. Accordingly, the intrinsic properties of viral structural proteins, in particular, the specific membrane domains to which they are targeted, might influence tetherin sensitivity. Nevertheless, these data show that tetherin can act in a very broadly specific way, as is characteristic of innate immune effector activities. An understanding of precisely how tetherin and its antagonists function could therefore provide therapeutic opportunities for a variety of viral infections.