The many mechanisms of viral membrane fusion proteins.

Every enveloped virus fuses its membrane with a host cell membrane, thereby releasing its genome into the cytoplasm and initiating the viral replication cycle. In each case, one or a small set of viral surface transmembrane glycoproteins mediates fusion. Viral fusion proteins vary in their mode of activation and in structural class. These features combine to yield many different fusion mechanisms. Despite their differences, common principles for how fusion proteins function are emerging: In response to an activating trigger, the metastable fusion protein converts to an extended, in some cases rodlike structure, which inserts into the target membrane via its fusion peptide. A subsequent conformational change causes the fusion protein to fold back upon itself, thereby bringing its fusion peptide and its transmembrane domain-and their attached target and viral membranes-into intimate contact. Fusion ensues as the initial lipid stalk progresses through local hemifusion, and then opening and enlargement of a fusion pore. Here we review recent advances in our understanding of how fusion proteins are activated, how fusion proteins change conformation during fusion, and what is happening to the lipids during fusion. We also briefly discuss the therapeutic potential of fusion inhibitors in treating viral infections.

Critical role for the cysteines flanking the internal fusion peptide of avian sarcoma/leukosis virus envelope glycoprotein.

The transmembrane subunit (TM) of the envelope glycoprotein (Env) of the oncovirus avian sarcoma/leukosis virus (ASLV) contains an internal fusion peptide flanked by two cysteines (C9 and C45). These cysteines, as well as an analogous pair in the Ebola virus GP glycoprotein, are predicted to be joined by a disulfide bond. To examine the importance of these cysteines, we mutated C9 and C45 in the ASLV subtype A Env (EnvA), individually and together, to serine. All of the mutant EnvAs formed trimers that were composed of the proteolytically processed surface (SU) and TM subunits. All mutant EnvAs were incorporated into murine leukemia virus pseudotyped virions and bound receptor with wild-type affinity. Nonetheless, all mutant EnvAs were significantly impaired ( approximately 1,000-fold) in their ability to support infectivity. They were also significantly impaired in their ability to mediate cell-cell fusion. Our data are consistent with a model in which the internal fusion peptide of ASLV-A EnvA exists as a loop that is stabilized by a disulfide bond at its base and in which this stabilized loop serves an important function during virus-cell fusion. The fusion peptide of the Ebola virus GP glycoprotein may conform to a similar structure.

The central proline of an internal viral fusion peptide serves two important roles.

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (beta-sheet), a tight reverse turn containing the proline, and a second region of order (alpha-helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.

Activation of a retroviral membrane fusion protein: soluble receptor-induced liposome binding of the ALSV envelope glycoprotein.

It is not known how membrane fusion proteins that function at neutral pH, for example the human immunodeficiency virus envelope (Env) glycoprotein and intracellular fusion machines, are activated for target bilayer binding. We have addressed this question using a soluble oligomeric form of an avian retroviral Env glycoprotein (API) and soluble forms of its receptor. Binding of soluble receptor to API induces API to bind to liposomes composed of phosphatidylcholine and cholesterol at neutral pH. Liposome binding only occurs at fusion permissive temperatures (T > 20 degrees C), is complete between 2 to 5 min at 37 degrees C, and is stable to high salt, carbonate, and urea. Liposome binding is mediated by the ectodomain of the transmembrane subunit of API, and a mutant with a Val to Glu substitution in the Env fusion peptide (located in the ectodomain of the transmembrane subunit) shows significantly reduced liposome binding. Moreover, under conditions of equivalent binding to API, a mutant receptor that does not support infection (Zingler, K., and J.A.T. Young. 1996. J. Virol. 70:7510-7516) does not induce significant liposome binding. Our results indicate that a highly specific interaction between an avian retroviral Env and its receptor activates the retroviral glycoprotein for target bilayer binding at neutral pH in much the same way as low pH activates the influenza hemagglutinin. Our findings are discussed in terms of the mechanisms of viral and cellular fusion proteins that function at neutral pH.

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres.

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

In vivo and in vitro association of hsc70 with polyomavirus capsid proteins.

Members of the 70-kDa family of cellular stress proteins assit in protein folding by preventing inappropriate intra- and intermolecular interactions during normal protein synthesis and transport and when cells are exposed to a variety of environmental stresses. During infection of A31 mouse fibroblasts with polyomavirus, the constitutive form of hsp70, hsc70, coimmunoprecipitated with all three viral capsid proteins (VP1, VP2, and VP3). In addition, the subcellular location of hsc70 changed from cytoplasmic to nuclear late in polyomavirus infection, coincident with the nuclear localization of the viral capsid proteins. VP1 and VP2 expressed in Sf9 insect cells with recombinant baculovirus vectors also coimmunoprecipitated with an hsp70-like protein, and VP1 expressed in Escherichia coli coimmunoprecipitated with the hsp70 homolog DnaK. Capsid proteins expressed by in vitro translation coimmunoprecipitated with the hsc70 protein present in the reticulocyte translation extract. Therefore, the polyomavirus capsid proteins associate with hsc70 during virus infection as well as in recombinant protein expression systems. This association may play a role in preventing the premature assembly of capsids in the cytosol and/or in facilitating the nuclear transport of capsid protein complexes.

Polyomavirus VP1 phosphorylation: coexpression with the VP2 capsid protein modulates VP1 phosphorylation in Sf9 insect cells.

The polyomavirus virion has an outer capsid comprised of 72 pentamers of the VP1 protein associated with the minor virion proteins, VP2 and VP3, and the viral minichromosome. To investigate the interaction between VP1 and VP2/VP3, we mapped VP1 phosphorylation sites and assayed VP1 recognition by anti-peptide antibodies after coexpression of VP1 with VP2 or VP3 by using recombinant baculovirus vectors. VP1, expressed either alone or with VP3, was phosphorylated on serine residues, which are not modified during polyomavirus infection of mouse cells. When VP1 was coexpressed with VP2, the nonphysiologic serine phosphorylation of VP1 was decreased, and a tryptic peptide containing Thr-63, a site modified during virus infection of mouse cells, was phosphorylated. An anti-peptide antibody directed against the VP1 BC loop domain containing Thr-63 recognized VP1 expressed alone but not VP1 coexpressed with VP2 or VP3. The change in phosphorylation resulting from coexpression of two structural proteins identifies the potential of the baculovirus system for studying protein-protein interactions and defines a functional role for the VP1-VP2 interaction.

Expression of the polyomavirus VP2 and VP3 proteins in insect cells: coexpression with the major capsid protein VP1 alters VP2/VP3 subcellular localization.

During polyomavirus infection the capsid proteins are synthesized in the cytoplasm and transported into the nucleus were virion assembly occurs. Expression of the major capsid protein VP1 in Sf9 insect cells results in the accumulation of capsid-like particles in the nucleus, independent of the presence of the minor capsid proteins VP2 and VP3 or the viral DNA (Montross et al., J. Virol. 65, 4991-4998, 1991). Sf9 cells infected with baculovirus vectors expressing the polyomavirus minor capsid proteins VP2 and VP3 were examined. VP2 was myristylated in Sf9 cells, as seen during polyomavirus infection of mouse cells. Immunoprecipitation of lysates from co-infected cells demonstrated an association between VP1 and VP2. As determined by immunogold electron microscopy, when expressed alone VP2 was associated with membrane structures in the cytoplasm and VP3 was diffusely localized in the cytoplasm. When co-infected with a VP1 expressing baculovirus, both VP2 and VP3 became predominantly localized to the nucleus in association with capsid-like structures. Thus, the polyomavirus capsid proteins interact in vivo and alter their subcellular localization as a consequence.