Entry into a host cell is a critical initiating event in the infectious cycle of a virus. Our understanding of this event is crucial both for future hopes of developing therapeutics to block the entry of viral pathogens and for the ability to exploit viruses as vectors for targeted gene delivery. It is clear that enveloped viruses have evolved to use a variety of host molecules to mediate their attachment to the cell and use different physical clues to activate the fusogenic capacities of their glycoproteins. Despite these differences, there is ever-growing evidence that many viral glycoproteins from highly divergent viruses share common architectural and structural motifs (8
), suggesting that these viruses may exploit common mechanisms to mediate membrane fusion. EnvA, the envelope glycoprotein of ASLV, a prototypical retrovirus, contains many of these motifs (21
). Thus, our increased appreciation of the dynamic interactions and structural changes that occur during EnvA-mediated entry may have implications for our understanding of entry by other retroviruses, such as HIV.
Here, we examined the effects of mutations within the RBD on early events in ASLV entry to delineate the role of the RBD in receptor-triggered activation of EnvA. Mutations within the RBD do not appear to disrupt the global structure or oligomeric status of EnvA. Previous analysis of these three RBD mutants had demonstrated that they are processed and incorporated into viral particles similarly to wild-type EnvA (42
), indicating that there are no gross disruptions in the overall structure of the glycoprotein. Density gradient centrifugation demonstrates that all three mutants sediment at a position comparable to that of wild-type EnvA PI, indicating that these mutations did not alter the formation or stability of the glycoprotein trimer. Therefore, the biochemical and functional properties of these RBD mutants do not appear to be due to altered stability of the glycoprotein oligomer, and as might be expected, the RBD does not appear to play a role in EnvA oligomerization.
The abilities of the RBD mutants to respond to receptor binding appropriately, inducing structural rearrangements in the SU and TM subunits, were variable and did not correlate with the abilities of the proteins to bind receptor. Both M20 PI and M28 PI demonstrated enhanced sensitivity to protease in the presence of high levels of sTva (Table ), consistent with receptor-induced structural rearrangements within the SU subunits of these envelope proteins. There were no detectable differences in the rate or thermal profile of Tva-induced protease sensitivity for M20 PI or M28 PI compared to those of wild-type EnvA PI (data not shown.) These two RBD mutants, therefore, were competent to undergo the initial structural changes in SU induced by Tva binding.
In contrast, biochemical analysis of M21 PI established that this mutant was sensitive to proteolytic digestion independent of Tva binding, suggesting an alteration in the native structure of SU. This phenotype initially suggested the possibility that the protein no longer required Tva in order to undergo structural rearrangements and existed in a preactivated state. However, analysis by liposome flotation assays demonstrated that M21 PI failed to bind the target membrane despite prolonged incubation with or without excess receptor (data not shown). The inability of M21 PI to associate with liposomes favors the hypothesis that the basic residues at positions 223 and 224 (Table ) played a role in stabilizing the structure of SU or of EnvA as a whole. This is consistent with previous mutagenesis studies, which indicated that mutations at only positions 223 and 224 of SU had negligible effects on receptor binding but appeared to accentuate the effects of mutations at residues 213 and 227 (42
). These results suggested that residues 223 and 224 participate in receptor binding by maintaining the tertiary structure of the RBD and/or by directly stabilizing the interactions with receptor. The constitutive sensitivity of M21 PI to thermolysin, coupled with the inability of this mutant to form a complex with target membranes, is consistent with these mutations destabilizing the structure of SU. Analysis of the protease-sensitive phenotype of additional mutants containing alterations at residues 223 and 224 is required to confirm this hypothesis.
The pH-dependent glycoprotein of influenza, HA, can be converted to a fusogenic structure at neutral pH through destabilization of the protein with heat or chemicals, supporting a “metastable model” for the native conformation of the fusion protein (7
). The recent work of Carr et al. suggests that low pH is not a specific requirement for fusion activity and that activation of HA involves general destabilization of the native, metastable conformation and conversion to a more thermodynamically stable, active state (7
). Receptor-induced changes in EnvA are biochemically distinct from the changes induced by general destabilization. M21 PI appears to represent a destabilized form of the SU subunit of EnvA PI, yet under all conditions analyzed this protein was unable to convert to an active, membrane-binding state. Unlike the effects of mutant M21 PI on protease sensitivity, heat destabilization of wild-type EnvA PI does not result in the formation of SUtherm
in protease sensitivity assays. Rather, a smaller proteolytic product of approximately 19 kDa is formed when EnvA PI is exposed to temperatures greater than 50°C (data not shown). In the absence of sTva, this heat-destabilized EnvA PI protein does not bind liposomal membranes to an appreciable degree (unpublished data), further suggesting that it is not in an active conformation. While the native state of EnvA may also represent a metastable conformation, destabilization through mutagenesis or with heat appears insufficient for conversion to an active conformation. Coupled with the findings for M21 PI, these results suggest that specific interactions with the viral receptor are required for activation of EnvA, and this may represent another distinction between the activation of pH-independent and pH-dependent glycoproteins.
While mutants M20 PI and M28 PI exhibited wild-type responses to sTva binding in protease sensitivity assays, the mutants demonstrated minimal or no detectable conversion to a membrane-binding conformation in liposome flotation assays. Membrane association is mediated through the TM subunit (27
); therefore, these results suggest that the mutants M20 PI and M28 PI are blocked at a step or steps prior to exposure of hydrophobic regions within TM. The observed block to activation was not alleviated by increasing the temperature of the reaction, suggesting it was not a result of alterations in the thermodynamic activation profile of EnvA and could not be overcome by destabilizing the protein. The inefficient and slow rate of membrane binding seen with M20 PI indicates that this mutant dissociates Tva-triggered conformational changes in SU from changes in TM. The decreased sTva-binding capacity of M20 PI may contribute in part to the observed delay in membrane association. However, the significant increase in the sensitivity of SU to protease suggests that sTva binding is sufficiently avid to induce a relatively rapid conformational change in M20 PI. We cannot formally exclude the possibility that suboptimal binding is sufficient for the initial conformational changes in SU while wild-type receptor-binding affinity is necessary for acquisition of a fusogenic state. However, it is clear from studies of retroviral envelope proteins that high-affinity binding is not a prerequisite for viral entry. There are numerous examples of mutations within ASLV, murine leukemia virus, and HIV envelope proteins that diminish their capacities to bind their respective host cell receptors, yet these mutations have no demonstrable effects on envelope-mediated membrane fusion or infection (33
). Therefore, we favor a model in which the RBD of ASLV envelope is intimately involved in transducing the activation signal from the receptor-binding site to the TM subunit.
The requirement for an intact RBD during the activation of EnvA is more dramatically illustrated by biochemical analysis of mutant M28 PI. Despite the ability of M28 PI to efficiently bind to sTva and undergo conformational rearrangements, this RDB mutant failed to bind to target membranes under any circumstances tested. In vitro, M28 PI demonstrates a complete uncoupling of receptor-triggered changes in the SU and TM subunits, again supporting the role of the RBD in transduction of the activation signal to the fusogenic TM subunit. The biochemical properties of M28 PI suggest that a basic residue at position 213 of SU is critical for transmission of the activation signal to TM. Thus, these two RBD mutants, M20 and M28, clearly demonstrate dissociation of the Tva-induced changes in the receptor-binding subunit from changes in the fusogenic subunit and confirm an essential role of the RBD for full activation of this pH-independent virus.
Acquisition of protease sensitivity and membrane association represent changes on the same pathway (Fig. ); therefore, identification of mutants that uncouple these events favors a sequential model in which receptor binding induces changes in the structure of SU prior to membrane binding via TM (Fig. ). The sequential nature of the activation of EnvA is analogous, in part, to the early events believed to occur when the glycoprotein of HIV type 1 (HIV-1) interacts with the host cell. For HIV-1, the SU subunit (gp120) initially binds the primary receptor, CD4 (1
), leading to structural changes that enable the glycoprotein to utilize a coreceptor molecule, CXCR4 or CCR5 (9
). Similar to sTva-triggered changes in EnvA, CD4 binding exposes cryptic protease sites in gp120 (44
). However, CD4-induced changes, which also include structural rearrangements within SU and frequently shedding of this subunit (3
), are insufficient for virus entry (34
). Secondary conformational changes are required for the glycoprotein to expose hydrophobic residues and acquire a fusogenic potential, and these changes occur following coreceptor binding (29
). In contrast to HIV-1 Env, EnvA requires a single cellular receptor, Tva, to achieve an active conformation. Here, we have demonstrated that mutations within the RBD are able to dissociate the first Tva-induced changes in SU from the secondary changes needed to insert into the target cell membrane (Fig. ). The dissociation of early steps in the activation pathway supports a role for the RBD in coupling the activation signal induced by receptor binding from the SU to the TM subunit. This suggests that communication between the two subunits may be critical for efficient receptor-triggered activation of pH-independent viral entry.
FIG. 8 Model of early events in EnvA-mediated entry. Through the RBD in SU, trimeric EnvA binds multiple Tva molecules on the surfaces of avian cells (a). This binding triggers structural rearrangements in the SU subunit, which likely include lateral dissociation (more ...)
The functional defect defined by the RBD mutant M28 suggests that there is cross talk between the SU and TM subunits during receptor-triggered activation of EnvA, with the RBD in SU playing a central role in coupling the activation signal. It appears that mutations in the viral envelope proteins which produce phenotypes similar to that of M28 (i.e., thus mainten wild-type receptor binding yet are defective for entry) are very rare. Obvious exceptions are the HIV-1 gp120 mutants, which maintain wild-type CD4 binding but have a restricted cellular tropism, likely representing changes in the interaction of gp120 with the coreceptor molecules (CXCR4 and CCR5) (10
). Nonetheless, there is indirect evidence of communication between the RBD within gp120 and the TM subunit of HIV-1, gp41. Resistance to neutralization by antibodies to the CD4-binding site maps to a single substitution in gp41 (582 A/T) (30
). This substitution dramatically reduces the ability of antibodies to bind the CD4-binding site within gp120 compared to their ability to bind that of the parental virus, HXB2 (49
). The neutralization-resistant mutant, HXB2thr582, has a greater propensity to form syncytia in tissue culture than HXB2, suggesting that the mutant envelope protein may be more fusogenic (49
). This indicates that the TM subunit of HIV can modulate the structure of the receptor-binding site; moreover, it suggests that interactions between the receptor-binding site in gp120 and the TM subunit are functionally important for initiating HIV entry into cells. Analysis of M28 PI demonstrates the first biochemical characterization of a receptor-binding site mutant which uncouples these early events in viral entry and further supports the importance of functional interactions between the receptor-binding site in SU and the TM subunit during pH-independent retrovirus entry.
Membrane fusion is an important and ubiquitous cellular process that plays a role in diverse biological events ranging from fertilization to neurotransmission. Protein-protein interactions appear to play a critical role in the regulation of vesicle fusion (28
). Recent work by Weber et al. suggests that interactions between two proteins, vesicle SNARE and target SNARE, are sufficient to mediate membrane fusion in vitro and, thus, that these SNARE proteins represent a minimal machinery of intracellular vesicle fusion (51
). The regulation of protein fusogenicity by specific protein-protein interactions may therefore represent a common means of modulating membrane fusion. The interactions between EnvA and Tva represent a simple viral model of a protein-regulated fusion machine. Thus, a detailed understanding of this system may further our understanding of virus entry, as well as cellular membrane fusion in general.