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Journal of Virology, December 2006, p. 11982-11990, Vol. 80, No. 24
0022-538X/06/$08.00+0     doi:10.1128/JVI.01318-06

Stoichiometry of Murine Leukemia Virus Envelope Protein-Mediated Fusion and Its Neutralization

Wu Ou and Jonathan Silver^*

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Received 22 June 2006/ Accepted 29 September 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection by enveloped viruses starts with fusion between viral and cellular membranes, which is mediated by envelope glycoproteins (Envs), usually organized as oligomers. For murine leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), the Env proteins trimerize and become glycosylated in the endoplasmic reticulum en route to the Golgi body. In the Golgi body, sugars are modified and Env is proteolytically cleaved into a surface protein species designated SU and a transmembrane protein designated TM, which are linked through a disulfide bond in MLV (7, 8, 14). Following transfer to the cell surface and incorporation into virus particles, the cytoplasmic tail of the MLV Env is cleaved by the viral protease, a step that is necessary to activate the fusogenic potential of MLV Env (12, 30, 31). Binding of SU to its cellular receptor(s) induces conformational changes in SU that expose the fusion machinery of TM; TM then pulls viral and cellular membranes together by forming a trimer of hairpin-like structures common to many different viral Envs (6, 8, 10, 11, 39-41).

While MLV and HIV-1 Envs function as trimers, not all of the molecules in a trimer need to be functional. Thus, some Env mutants form functional heterotrimers with wild-type Env (44), and some Envs with lethal mutations in SU can complement Envs with lethal mutations in TM (35, 47). Two kinds of heterotrimers may be formed, which can be designated X2Y1 and X1Y2, where X and Y stand for the different monomers and 1 and 2 stand for the number of monomers of each type in a trimer. A recent study found that trimers with one but not two mutant monomers were functional (44). Whether one or both forms of heterotrimer are functional is relevant to a detailed understanding of the mechanism by which Env induces membrane fusion.

For viruses such as HIV-1 that can establish chronic infections and "hide" inside cells, an effective vaccine may have to generate neutralizing antibodies capable of inactivating all incoming virus. While the feasibility of such "sterilizing" immunity is controversial, sterilizing immunity has been documented in a few cases (9, 19). Most antibodies elicited by HIV-1 are nonneutralizing; however, a few broadly reactive, potent neutralizing antibodies against HIV-1 have been described elsewhere (2, 3, 5, 18, 20, 21, 24, 33, 36, 38, 45, 46). Among them, 2F5 has been extensively studied. 2F5 targets a linear epitope in the membrane-proximal region of HIV-1 TM (5, 22-24, 29, 49, 50). Understanding how these antibodies neutralize HIV-1 infection could be important for designing new AIDS vaccines.

There are many theories concerning mechanisms of virus neutralization by antibodies, including steric blocking of interaction with receptors and blocking of subsequent conformational changes. Different antibodies may have different mechanisms. Stoichiometry of antibody binding for neutralization has been extensively studied, but debate about how many antibody molecules need to bind per virion for neutralization continues (4, 16, 17, 37, 43).

In this study, we address questions concerning the stoichiometry of MLV Env-mediated fusion and its inhibition by antibody, including how many functional SU or TM molecules are required for an Env trimer to be fusion competent; how many antibody molecules are needed to block the function of one trimer; whether the stoichiometry of neutralization is the same for an epitope in SU as in TM; and whether antibodies can inactivate a trimer by binding only to nonfunctional SU or TM in the trimer and, if so, whether they are as efficient in this case as when they bind to functional molecules.

Our strategy is based on MLV Env complementation (47, 48) and our previous work showing that 2F5 blocks membrane fusion mediated by MLV Envs with a 2F5 epitope inserted in either SU or TM (25). We placed complementing point mutations in the receptor binding site in SU and a site in TM involved in fusion, in Envs marked with 2F5 or hemagglutinin (HA) epitopes. We cotransfected pairs of Envs to produce complementing heterotrimers with a 2F5 epitope in mutant SU, wild-type SU, wild-type TM, or mutant TM (Fig. 1A, B, C, and D, respectively). For convenience, these Env complementations are designated pairs A, B, C, and D throughout the paper. We investigated the effects of cotransfection on Env synthesis, transport to the cell surface, fusion efficiency, and neutralization by 2F5 antibody.

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FIG. 1. Schematic diagram of Env pairs tested indicating relative positions of point mutations (D84K and L493V) and epitopes (2F5 and HA) inserted in SU and TM.

Top Abstract Introduction Materials and Methods Results Discussion References

 
MLV Env constructs. MLV Env constructs with the 2F5 epitope in SU or TM or with an HA epitope in SU were previously described (25). They encode fusion-active forms of Env that contain a stop codon at the normal p12e cleavage site in the cytoplasmic tail of TM. We used overlapping PCR to introduce D84K and L493V mutations into SU and TM, respectively. The primers used to mutate D84 to K were 5'-GCAGCCCAGGCTGTTCCAGAAAGTGCGAAGAACCTTTAACC-3' and 5'-GGTTAAAGGTTCTTCGCACTTTCTGGAACAGCCTGGGCTGC-3'. Primers for L493V were 5'-GGTTGAAAAATCAATCTCTAACGTTGAAAAGTCTCTCACTTCCC-3'and 5'-GGGAAGTGAGAGACTTTTCAACGTTAGAGATTGATTTTTCAACC-3'. Mutated nucleotides are highlighted by boldface italic. Each construct was verified by sequencing.

Antibody and cell lines. Human anti-HIV-1 monoclonal antibody 2F5 was from Polymun Scientific (Vienna, Austria; catalog no. AB001). HEK293 and U₂OSLucCAT target cells (28) were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U penicillin, and 100 U streptomycin/ml. All cells were kept at 37°C in a 5% CO₂ humidified incubator.

MLV Env expression. HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen) to express MLV Envs. Surface proteins were biotinylated 24 h after transfection using a membrane-impermeant biotin reagent from Pierce (catalog no. 20338). An aliquot of total cell lysate and proteins purified using streptavidin beads (Pierce; catalog no. 29200) was analyzed by Western blotting to check the total and surface expression level of each Env, respectively. The membrane was first blotted with anti-HA antibody, followed by horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G antibody, and developed with Pierce SuperSignal ECL reagents (catalog no. 1856136). Then the membrane was stripped using Pierce Stripping Buffer (catalog no. 21059) and reblotted with 2F5 and HRP-conjugated anti-human immunoglobulin G antibodies. To check loading and biotinylation of surface proteins, the membrane was stripped again and blotted with HRP-conjugated streptavidin (Pierce; catalog no. 21126). More details can be found in previous reports (25-27).

MLV Env complementation and neutralization. We used a quantitative cell fusion assay (28) to assess MLV Env complementation and neutralization. Briefly, effector cells (HEK293) were seeded in a six-well plate at a density that resulted in their being 90 to 95% confluent the next day. Cells were cotransfected with pTet-Off (Clontech, Mountain View, CA) plus various ratios of MLV Env expression vectors. A total of 4 µg of plasmid DNA and 10 µl Lipofectamine 2000 (Invitrogen, Carlsbad, CA) were used for each well, following the manufacturer's protocol. Twenty-four hours later, the cotransfected cells were trypsinized and preincubated with or without 2F5 antibody at 4°C for 30 min. Then U₂OSLucCAT target cells were added and cells were cultured in triplicate wells at 37°C. Antibody was left in the coculture medium. Luciferase activity was assayed about 16 h after coculture using the Luciferase Assay System from Promega (Madison, WI) and a Victor 3 luminometer (Perkin-Elmer, Boston, MA).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Env constructs for complementation. Moloney MLV Env constructs expressing Env with a 2F5 epitope inserted in SU or TM or an HA epitope in SU have been described previously (25). Briefly, an extended 2F5 epitope (or an HA epitope where it applies) was inserted at a unique SgrAI site in the SU gene, in a region that encodes a proline-rich segment previously shown to tolerate small insertions (15, 34, 42), or inserted 14 amino acids upstream of the transmembrane domain of TM, which is the location of the natural 2F5 epitope in HIV-1. Cells expressing these Envs specifically fuse with target cells expressing the ecotropic MLV receptor mCAT1 (mouse cationic amino acid transporter 1) (1). Fusion mediated by Envs bearing the 2F5 epitope can be neutralized by 2F5 antibody (25). For inactivating mutations in SU and TM, we chose a point mutation in SU that blocks receptor binding (D84K) and a point mutation in a heptad repeat region of TM reported to inhibit fusion per se (L493V). These mutations were reported to be efficiently expressed and incorporated into virus particles, to abrogate viral infection, and to complement each other when cotransfected (47, 48). We introduced these mutations into the SU or TM of our 2F5 epitope-tagged Moloney MLV Env constructs (Fig. 1). The Env names specify the modifications: for example, SU(D84K/HA)TM(2F5) designates an Env with a D84K mutation and an HA epitope in the SU and a 2F5 epitope in the TM.

Env expression. Total Env was evaluated by Western blot analysis of cell lysates (Fig. 2A). Surface Env was measured by biotinylating cell surface proteins with a membrane-impermeant reagent, followed by purification with streptavidin beads and Western blot analysis (Fig. 2C). Each Env was tagged with an HA and/or a 2F5 epitope. For both total and surface expression, we did one Western blot assay with anti-HA antibody (Fig. 2A and C, upper panels) and another with 2F5 antibody (Fig. 2A and C, lower panels), which facilitated comparison of each Env's expression level when it was transfected alone with that when it was cotransfected. Lysates from positive-control cells [transfected with SU(HA) Env] and negative-control cells (mock-transfected cells) are shown in lanes 11 and 12, respectively. Envs SU(D84K/HA) and SU(D84K/2F5) were well expressed and proteolytically processed (Fig. 2A, lanes 1 and 2). Both were transported to the cell surface, but less 2F5 Env was detected on the surface than HA Env (Fig. 2C, lanes 1 and 2). Env SU(D84K/HA)TM(2F5) showed a slight reduction in total cellular Env with normal proteolytic processing (Fig. 2A, lane 3) but was undetectable on the cell surface (Fig. 2C, lane 3). The 2F5 antibody detected a small amount of this SU-TM Env precursor in the cell lysate but no proteolytically processed SU, as expected for an Env with the 2F5 epitope in TM (Fig. 2A, lane 3, lower panel). Env SU(HA)TM(L493V) showed slightly reduced total Env but normal proteolytic processing (Fig. 2A, lane 4); Env SU(2F5)TM(L493V) showed slightly reduced total Env that was poorly proteolytically processed (Fig. 2A, lane 5); Env SU(HA)TM(L493V/2F5) showed significantly reduced total Env that was poorly proteolytically processed (Fig. 2A, lane 6). Surface expression was not detectable for any Env with the L493V mutation (Fig. 2C, lanes 4 to 6). Background bands showed that approximately equal amounts of total lysates were loaded (Fig. 2B). Streptavidin-purified surface proteins blotted with streptavidin-horseradish peroxidase showed that biotinylation, purification, and loading were approximately equal for all samples (Fig. 2D).

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FIG. 2. Western blot of total cell lysates (A and B) and surface-biotinylated proteins (C and D) in HEK293 cells transfected with Env expression vectors. Point mutations D84K ("D/K") and L493V ("L/V") and epitopes (HA and 2F5) carried by each Env are indicated below each lane for single transfections (lanes 1 to 6 and 11) and double transfections (lanes 7 to 10). Lane 12 shows a mock-transfection control. Upper panels in panels A and C were probed with anti-HA, and lower panels were probed with 2F5 antibody. gp85 is the Env precursor before proteolytic cleavage; gp70 is SU. Panel B shows the background band in the anti-HA blot assay as a loading control. Panel D shows blot C stripped and reprobed with streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal in all lanes. Results are representative of two experiments.

The fact that proteolytic processing and transport to the cell surface were altered for many combinations of point mutations/epitope insertions was not expected given prior reports that the point mutations did not affect Env expression and suggests that the point mutations (especially L493V) and epitope insertions have combined effects on protein folding.

Cotransfection of Envs at a 1:1 DNA ratio (the amount of each Env plasmid being half that when transfected alone) led in most cases to increased total and cell surface Env (Fig. 2A and C, lanes 7 to 10). We infer that the increased Env species are heterotrimers, since stability and trafficking of homotrimers are unlikely to be influenced by nonassociated Env molecules. Increased total and surface Env is likely due to altered folding of Env species when heterotrimerized versus homotrimerized. In the cotransfections where one SU carried an HA epitope and the other a 2F5 epitope (pairs A and B), the presence of both Env species on the cell surface (Fig. 2C, lanes 7 and 8) is consistent with most cell surface trimers being heterotrimers.

Cell fusion. A quantitative cell fusion assay which we previously developed (28) was used to assess each Env's fusion activity. Briefly, effector cells (HEK293) were transfected with an Env expression vector plus a vector (pTet-Off) that encodes the transcriptional transactivator tTA. Twenty-four hours later, the effector cells were cocultivated with target human osteosarcoma cells (U₂OS) that stably express the MLV receptor mCAT1 and a luciferase reporter gene under the control of a promoter (TRE, tetracycline response element) that is activated by tTA following fusion. Luciferase activity was assayed about 16 h after coculture and is expressed in relative light units (RLU) detected by a luminometer. We previously showed that our cell fusion assay is quite sensitive in that it detects Env-mediated fusion in situations of suboptimal Env expression when surface Env is undetectable by biotinylation-Western blotting (25).

Effector cells transfected with the positive-control Env SU(HA) gave 35,000 RLU of luciferase activity in the fusion assay (Fig. 3, bar 11), compared to 20 RLU for mock-transfected cells (Fig. 3, bar 12). Cells transfected with Envs carrying the D84K mutation were inactive in the fusion assay (Fig. 3, bars 1 to 3). This is expected since the D84K mutation abrogates receptor binding (47, 48). Cell fusion mediated by L493V mutant Envs was 7% that of the positive-control Env but clearly above background (Fig. 3, bars 4 to 6). The fact that the L493V mutation did not completely abrogate fusion in our cell fusion assay contrasts with published results obtained using a virus infection assay (47). The cell fusion assay may be more sensitive than the virus infection assay because cells have a surface area roughly 10,000 times larger than that of viruses. At equal Env surface density, there would be many more Env molecules per cell than per virus, and in situations where the fusogenicity of individual Env molecules is reduced by mutation, this increased number of Env molecules may be crucial for fusion.

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FIG. 3. Results of cell fusion assay. Indicator cells expressing ecotropic MLV receptor plus luciferase inducible by tTA were cocultured in triplicate with HEK293 cells transfected with expression vectors for tTA plus Env genes indicated below each lane (samples in same order as in Fig. 2; lane 12, no-Env control). Cell lysates were assayed for luciferase activity after 16 h of coculture. Mean luciferase activity is indicated above each bar; error bars show standard deviations. Results are representative of four or five experiments.

Cotransfection of mutant Envs led to increased fusion (Fig. 3, bars 7 to 10), most notably in the case of Env pairs B and C, where fusion was more than 15 times higher than with either mutant alone. Fusion could be due to heterotrimers or L493V homotrimers, but not D84K homotrimers. We can estimate the contribution from L493V homotrimers under various assumptions. If L493V monomers randomly associate with other monomers, then, since only half as much L493V-encoding DNA (0.6 µg) was transfected in the cotransfection, one expects (1/2)³ = 1/8 as much L493 homotrimer. Random association is consistent with equal intensity of HA and 2F5 species for Env pairs A and B in Fig. 2C. If fusion is proportional to the amount of cell surface Env, then the amount of fusion due to L493V homotrimers should be 1/8 as much after cotransfection as after single transfection. To test the proportionality assumption, we measured fusion as a function of the amount of Env DNA transfected, keeping total DNA constant by addition of empty vector. Fusion decreased slightly faster than proportionally with serial twofold dilutions of Env DNA over the range of 1.2 µg to 0.15 µg. For example, with wild-type Env, the mean luciferase activity decreased from 3,118, to 1,331, to 276 to 38 with a background of 10 RLU. If we take proportional decrease as a conservative assumption, then we would estimate for Env pair B that roughly (149 x 1/8) = 19 RLU out of the total 3,203 RLU, or 0.6%, is from homotrimer-mediated fusion (Fig. 3, bars 5 and 8). For Env pairs A, C, and D this argument leads to estimates that 4%, 0.8%, and 4% of the fusion is due to homotrimers, respectively. Even if the assumptions of the simple model are not quantitatively accurate, we conclude that most of the fusion seen following cotransfection is likely due to heterotrimers.

Inhibition of fusion by 2F5 antibody. To test for inhibition of fusion, effector cells were transfected in the same way as for Fig. 3. Twenty-four hours later, effector cells were trypsinized, preincubated with or without 2F5 antibody for 30 min at 4°C, and then cocultured with target cells. The antibody was left in the medium during coculture. Luciferase activity was assayed about 16 h after coculture and normalized to 100% for samples that had no 2F5 antibody present. 2F5 antibody inhibited fusion in a dose-dependent fashion for all Env pairs containing a 2F5 epitope in one of the Envs (Fig. 4, curves A to D), but it did not inhibit fusion mediated by Env lacking a 2F5 epitope (curve E), as expected. Fusion inhibition was seen at lower antibody concentrations (lower 50% inhibitory concentration) when the 2F5 epitope was in SU (curves A and B) than when it was in TM (curves C and D), as previously shown for singly transfected Envs (25). It is striking that 2F5 antibody inhibited fusion even when it could bind only to nonfunctional SU (curve A) or mutated TM (curve D). This shows that neutralizing antibody bound to one molecule (SU or TM) can have a significant inhibitory effect on the function of other molecules in the same trimer. We refer to this as inhibition "in trans."

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FIG. 4. Inhibition of cell fusion by 2F5 antibody. Cell fusion assays were performed as for Fig. 3 in the presence of increasing concentrations of 2F5 antibody. Results are normalized to the amount of luciferase activity in the absence of antibody. Env genes transfected are shown below the curve designations A to E. Results are representative of three experiments.

Effect of DNA transfection ratio on fusion and inhibition of fusion. When two different Env genes, say X and Y, are cotransfected, two kinds of heterotrimers can result: X1Y2 and X2Y1, where 1 and 2 refer to the number of monomers of a given type in the trimer. To test for possible differences between the two kinds of heterotrimers in fusion function or susceptibility to inhibition by 2F5, we varied the ratio of cotransfected Env DNAs from 10:1 to 1:10, keeping the total amount of transfected DNA constant.

The curves in the upper panels of Fig. 5 show that the amount of fusion in the absence of antibody was only mildly sensitive to the ratio of the two DNAs, varying at most by a factor of 3. If only one of the two species of heterotrimer were functional (say X2Y1), the expected variation in fusion would be much greater. For example, in the model described in Tables 1 and 2, the amount of fusion induced by X2Y1 or X1Y2 heterotrimer would vary by a factor of 21 (0.42/0.02) as the DNA transfection ratio varied from 10:1 to 1:10, whereas the amount of fusion induced by both heterotrimer species together would vary by only a factor of 3 (0.75/0.25). Thus, the data are more consistent with the model in which both heterotrimer species are functional.

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FIG. 5. Effects of varying the ratio of cotransfected Env genes on inhibition by 2F5 antibody. Cell fusion assays were performed as for Fig. 4 except that the ratio of cotransfected Env DNAs was varied from 10:1 to 1:10. Upper panels show luciferase activity in the absence of antibody; lower panels show luciferase activity in the presence of indicated concentrations of 2F5 antibody, normalized to luciferase activity in the absence of antibody. Diagrams above each graph show the Env genes transfected, depicted as trimers with two monomers of one type and one of the other, depending on which monomer was transfected in excess on each side of the graph. Panels A to D correspond to Env pairs A to D in Fig. 1. Results are representative of two or three experiments.

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TABLE 1. Relative amounts of different trimer species as a function of DNA transfection ratio, assuming equal synthesis, transport, and random association of monomers X and Y

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TABLE 2. Relative amounts of fusion, assuming that fusion is proportional to the amount of fusion-competent trimers and only X2Y1 (I), only X1Y2 (II), or both heterotrimer species (III) are fusion competent

Strictly speaking, this argument assumes that the two types of heterotrimers are formed and transported to the cell surface equally efficiently. For Env pairs A and B, the approximately equal reactivity of anti-HA and 2F5 antibodies shown in the lower panel of Fig. 2 supports this assumption when the DNAs are transfected at a 1:1 ratio. For other ratios and Env pairs C and D, this assumption is untested.

The extent of inhibition of fusion by 2F5 antibody varied with Env DNA ratio for Env pairs A and D, and in each case inhibition was more extensive at DNA ratios where most of the heterotrimers would have two 2F5 epitopes (left side, lower panel of Fig. 5A; right side, lower panel of Fig. 5D). If only one heterotrimer species were functional, one would expect the 2F5 antibody to inhibit fusion to the same relative extent independently of the absolute amount of that heterotrimer. Apparent differential sensitivity to 2F5 antibody of the two types of heterotrimer provides additional evidence that both types of heterotrimer are functional. Since one type of heterotrimer has only one functional SU, this implies that only one molecule of SU needs to bind receptor for a trimer to contribute to fusion.

In contrast, inhibition of fusion by 2F5 antibody did not vary with Env DNA ratio in the case of Env pairs B and C (Fig. 5, lower panels). Since subtle differences in sensitivity to neutralization between heterotrimer species are more likely to be seen at an intermediate level of inhibition, we further tested the flatness of the neutralization curves for Env pairs B and C using 2 µg/ml and 50 µg/ml 2F5 antibody, respectively. The neutralization was about 50% for both, and the curves remained flat (data not shown). One possible explanation for the flatness of the neutralization curves for Env pairs B and C is that only one form of heterotrimer is functional. We do not think this is likely, because experiments varying the Env ratios implied that both forms of heterotrimers were functional (see above). An alternative explanation is that heterotrimers with either one or two 2F5 epitopes are both functional but neutralized by 2F5 antibody with equal efficiency. Since trimers with only a single 2F5 epitope are blocked, this implies that a single antibody molecule can block the function of a whole trimer. The inference that trimers with a single epitope were blocked is supported by the observation that high concentrations of antibody almost completely inhibited fusion when almost all heterotrimers should contain a single 2F5 epitope (Fig. 5B, lower panel, left side).

What might account for the sensitivity to Env ratio for pairs A and D but not B and C? In Env pairs A and D, the 2F5 epitope is in the nonfunctional (D84K) SU or poorly functional (L493V) TM, whereas in Env pairs B and C, the 2F5 epitope is in the functional SU or TM. A possible interpretation is that when 2F5 antibody binds only to nonfunctional or weakly functional subunits in a heterotrimer (i.e., when it acts in trans), it is more efficient at inhibiting function when two antibody molecules can bind or when one antibody molecule can bind two epitopes in the same trimer. In contrast, when the 2F5 antibody binds to the functional Env species in a heterotrimer, it blocks the heterotrimer function equally well whether one or two antibody molecules can bind. This result appeared to be true when the epitope was in SU (Fig. 5B) as well as TM (Fig. 5C).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanism of complementation is not yet clear, though it is usually considered in terms of SU and TM from different monomers being able to work together to induce fusion (32, 35, 47, 48). Our results support another mechanism that may contribute to complementation in some cases, namely, heterotrimerization increasing steady-state levels and transport to the plasma membrane of mutant Env species that misfold as homotrimers. One can view this as a chaperone function provided by complementing Envs via heterotrimerization. A dominant-negative form of heterotrimerization has been demonstrated with chimeric proteins containing an alpha-helical domain from HIV-1 or MLV TM that heterotrimerize with nascent, wild-type Env molecules and trap them in the endoplasmic reticulum (26, 27).

Several of our experiments provide evidence that both forms of heterotrimer are functional, which implies that one functional SU molecule in a heterotrimer is sufficient for the trimer to be functional. A recent paper based on quantifying viral infectivity as a function of the ratio of cotransfected wild-type HIV-1 Env to Envs with null mutations for receptor (or coreceptor) binding or TM function reached a different conclusion, namely, that at least two wild-type Env molecules are required for a trimer to be functional (44). The existence of complementation by itself argues that trimers do not need to have two wild-type monomers to function, since complementing Envs have either only one wild-type SU or only one wild-type TM. Yang et al. found no complementation in their system, but this contrasts with the results reported here as well as several other reports (32, 35, 47, 48). Previous reports on complementation do not distinguish whether one or both heterotrimers are functional. Thus, our finding that one wild-type SU appears to be sufficient to make a trimer functional is new. It should be kept in mind that the conclusion that only a single wild-type SU is required is restricted to the context of a heterotrimer. The receptor-binding mutant SUs in a heterotrimer could provide other functions, for example, by making crucial contacts with other monomers to provide structural integrity or transmit conformational changes to TM. Our data do not allow us to make the analogous argument that only one wild-type TM molecule is needed in a trimer because the L493V mutation we used unexpectedly had some fusion activity on its own.

Measuring the ability of antibody to neutralize heterotrimers carrying different epitopes allows inferences about the number of antibody molecules required to inactivate a trimer. We inferred that one 2F5 antibody molecule was sufficient when its epitope was in functional SU or TM because X2Y1 and X1Y2 species were neutralized with equal efficiency and neutralization occurred even at DNA ratios at which most heterotrimers would carry a single 2F5 epitope. The same conclusion has been reached by others (37, 43). We previously showed that the 2F5 antibody slightly inhibited but did not prevent virions with 2F5 epitopes in SU from binding to cells with the mCAT1 receptor (25). Therefore, we favor the idea that one antibody molecule can block fusion by interfering with conformational changes that occur after receptor binding.

Interestingly, we found that when the 2F5 epitope was carried on a nonfunctional or poorly functional subunit, fusion was more efficiently inactivated at DNA transfection ratios that favored formation of heterotrimers with two 2F5 epitopes (sloping curves, Fig. 5A and D, lower panels). This was not the case when the 2F5 epitope was carried on the functional subunit (flat curves, Fig. 5B and C, lower panels), which suggests a subtle difference in mode of action when the antibody acts in cis (inhibiting the function of the molecule to which it binds) versus trans. It is straightforward to imagine how binding to functional versus nonfunctional Env subunits could have different effects on conformational changes induced by receptor binding. Herrera et al. (13) recently reported that antibody binding to nonfunctional Env monomers did not inhibit fusion, but in their system the Env mutations were dominant negative, so trimers that bound antibody were nonfunctional, whereas in our system the trimers that bound antibody were functional.

Testing the effects of 2F5 antibody on virus neutralization would strengthen our experiments. However, the titer of virus derived from cotransfection of epitope-tagged mutant Envs was too low to permit this. We are attempting to overcome this problem and test the generality of our conclusions using other point mutations and epitopes.

 
We thank Jamie Spangler for careful reading and comments on the manuscript.

 
* Corresponding author. Mailing address: Building 4/Room 336, 4 Center Dr., Bethesda, MD 20892. Phone: (301) 496-3653. Fax: (301) 402-0226. E-mail: jsilver{at}nih.gov.

Published ahead of print on 11 October 2006.

Present address: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, December 2006, p. 11982-11990, Vol. 80, No. 24
0022-538X/06/$08.00+0     doi:10.1128/JVI.01318-06

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