Previously, our laboratory proposed a model in which ASLV enters cells by receptor-mediated endocytosis followed by low-pH-dependent fusion from an intracellular compartment (22
). In the present report, we further substantiate this model and provide evidence that ASLV-A virions associated with a transmembrane form of the TVA receptor are internalized more rapidly than those using a GPI-anchored form of the receptor. However, virions become resistant to inhibition by the lysosomotropic agent NH4
Cl at similar rates regardless of which TVA receptor is used to enter the cells. Since these NH4
Cl experiments measure the combined kinetics of the initial uptake and the subsequent trafficking of virus particles, the simplest model to explain these data would be that upon being internalized, virions associated with the GPI-anchored receptor are subsequently trafficked to a putative fusion compartment more rapidly than those using the transmembrane-anchored form.
Remarkably, most virions that entered cells expressing TVA800 remained highly infectious in the presence of NH4
Cl, whereas those entering TVA950-expressing cells did not. This difference between the two types of TVA receptors correlated with the lipid raft association of the majority of the TVA800 protein. These data suggest that association of the receptor with lipid rafts influences the intracellular fate of virions (Fig. ). The simplest model to explain these findings is that virions entering via TVA950 are trafficked via a degradative endocytic pathway that may involve multivesicular bodies, late endosomes, and lysosomes (10
). In contrast, virions entering cells via lipid raft-associated TVA800 may be endocytosed by a known, or as-yet-unknown, lipid raft-dependent endocytic pathway to a stable compartment. The loss of viral infectivity that was seen with NH4
Cl-treated TVA800-expressing cells (Fig. ) may be due either to some intrinsic property of this “stable” compartment or, instead, might be due to virion uptake by receptors that reside outside of lipid rafts, which perhaps traffic the virus to a degradative compartment similar to that accessed by TVA950. We presently cannot distinguish between these two possibilities.
FIG. 5. Model for ASLV-A entry via transmembrane and GPI-anchored receptors. Virions bound to TVA950 are predicted to be taken up into cells by endocytosis and, in the presence of NH4Cl, accumulate in a degradative compartment. In contrast, virions bound to TVA800 (more ...)
Consistent with our model, lipid raft-associated components seem to avoid the degradative endocytic pathway by being sorted into the recycling endosome, which contains a low level of endocytic proteases (9
). Furthermore, some GPI-anchored proteins, such as folate receptor and CD55, are endocytosed and trafficked through this recycling endosome (6
). Alternatively, TVA800-associated virions may use another lipid raft-dependent endocytic pathway, such as that used by CD59, which traffics directly between the plasma membrane and the Golgi apparatus (24
); that used by simian virus 40, a nonenveloped virus which enters cells via caveolae and accesses the endoplasmic reticulum via the caveosome, a novel sorting organelle (29
); or that used by the interleukin-2 receptor, which seems to be independent of these other endocytic pathways (16
). It is also possible that in the presence of NH4
Cl, virions bound to TVA800 and TVA950 are trafficked to different “domains” of the same intracellular compartment. In support of this idea, it was recently suggested that proteins targeted for degradation may be localized to the internal invaginations of late endosomes, while those found at the outer limiting membrane are poorly degradable (10
). The use of pathway-specific inhibitors as well as quantitative imaging techniques to track individual virions may help distinguish between these various models of viral entry.
By characterizing the entry pathway used by ASLV-A, we hope not only to gain a better understanding of the mechanism by which this retrovirus enters cells but also to obtain new insights into how lipid raft-associated cargo is taken up into cells and delivered to an acidic endosomal compartment. To date, there has not been much information available on how such cargo is trafficked to an acidic endosome. This information, in turn, may help us better understand the entry mechanisms of other viruses that use GPI-anchored receptors, such as Jaagsiekte sheep retrovirus (32
) and perhaps Ebola virus (5
). In addition, the fact that we can stably arrest ASLV-A infection in TVA800-expressing cells by using NH4
Cl is a novel finding, since low-pH-dependent viruses are generally unstable under these conditions, presumably because they have been delivered to a degradative compartment (20
). This feature of the ASLV system may allow for the isolation of virus-containing endosomes which, upon reacidification, may support virus-cell membrane fusion. If so, this system could allow for a detailed biochemical analysis of receptor priming, fusion, and of the poorly defined subsequent step of viral uncoating that leads to reverse transcription.