INTRODUCTION

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The Fv1 gene is one of a series of mouse genes, originally described in the early 1970s, that control the susceptibility of mice to MLV (14, 15). Many of the genes were found to modify target-cell proliferation or the immune response to the virus, but Fv1 acts in a cell-autonomous manner to restrict virus replication (22). The precise mechanism for restriction is unclear. It has been shown that viral replication is blocked at a stage after virus entry into the cell but before integration of newly synthesized viral DNA into the host genome (11, 20).

There are two major alleles of Fv1: Fv1ⁿ and Fv1^b. These alleles are able to block specific subclasses of MLV (8). Fv1ⁿ, found in NIH Swiss mice, is able to block replication of B-tropic MLV while allowing replication of N-tropic virus, and Fv1^b, found in BALB/c mice, acts vice versa. The block to infection is not absolute in vitro, but the number of infected cells is reduced by a factor of 50 to 1,000 (8). When expressed at natural levels, neither allele shows significant restriction of NB-tropic MLV. However, recent studies involving overexpression of Fv1^b indicate that its gene product can interact to a certain degree with both N- and NB-tropic virus (3). By contrast, the product of the n allele does not show such secondary effects (3).

Genetic studies have shown that the target for Fv1 restriction is the MLV CA protein (9, 21). Subsequent studies suggested that viral tropism is determined by a pair of adjacent amino acids, residues 109 and 110, in CA (6, 18). A more recent study has shown that the amino acid at position 110 appears to be the most important residue for N- and B-tropism (13). N-tropic MLVs have Arg at this position, and B-tropic MLVs have Glu. The determinants for NB-tropism have not been fully characterized.

The Fv1 gene was cloned a few years ago (2) and was found to have sequence similarity (60% identity over 1.3 kb) to families of human and murine endogenous retroviruses called HERV-L or MuERV-L, respectively (1, 2). Based on its position within the Gag gene of the element, Fv1 apparently encodes a CA-like protein. Gag proteins bind tightly to each other via interaction domains during virus assembly (16), and this suggests a possible mechanism for the way Fv1 acts on MLV CA (7). To date, however, there is no evidence for direct binding of Fv1 to CA, and the extremely low level of expression of Fv1 in vivo effectively precludes direct biochemical analysis.

One feature of CA proteins is the MHR (19, 28). This domain is characterized by three absolutely conserved residues, as well as one aromatic and two hydrophobic amino acids, with exact spacing between them (Q-X₃-E-X₄--O-X-R-O, where  is the aromatic amino acid and O indicates an aliphatic amino acid) (1). It is the only region of significant sequence homology between CA proteins of different retroviral genera (28). An MHR motif is present in all replication-competent retroviruses analyzed, except spumaviruses (25), and can also be identified in the Fv1 ORF product (1). The biological significance of the MHR is unknown, but its clear evolutionary conservation must presumably reflect an important function. Mutational analyses of the MHR of HIV-1 (17), RSV (5), and Mason-Pfizer Monkey virus (25) have shown that specific residues are required for particle assembly, maturation, and proper function of the viral core in the early stages of infection. The role of the MHR of Fv1 has not been determined.

To identify the regions of Fv1 necessary for activity and the determinants of restriction specificity, we have taken a genetic approach. We created several different types of Fv1 mutants by site-directed mutagenesis, including both N-and C-terminal deletions, internal deletions throughout the coding region, and point mutations in the putative MHR domain of Fv1 and at residues 358 and 399. Using a recently developed transient assay for Fv1 function (3), these mutants were then typed for restriction activity. This paper describes the activities of these mutants and the conclusions we can draw about the determinants involved in Fv1 restriction.

	MATERIALS AND METHODS

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Abbreviations. The following abbreviations are used: CMV, cytomegalovirus; MLV, murine leukemia virus; CA, the MLV capsid protein (p30); EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; MHR, major homology region; ORF, open reading frame; HIV-1, human immunodeficiency virus type 1; FACS, fluorescence-activated cell sorting; SDS, sodium dodecyl sulfide; PBS, phosphate-buffered saline; B-MLV, B-tropic MLV; N-MLV, N-tropic MLV; NB-MLV, NB-tropic MLV; RSV, Rous sarcoma virus.

Recombinant DNA. All recombinant DNA work was done by established techniques (23). The structure of each plasmid prepared was verified by restriction mapping and/or sequencing prior to use. All DNA preparations were purified on Qiagen columns prior to transfection.

Cells and viruses. Cells were cultivated in Dulbecco's modified Eagle medium containing 10% fetal calf serum and antibiotics. Viruses were generated by simultaneous CaPO₄-mediated transient transfections of 293T cells with three plasmids providing vector, gag-pol and env functions (3, 24). CMV promoter-driven expression was stimulated by incubation in Dulbecco's modified Eagle medium containing 10 mM sodium butyrate for 8 to 10 h. Virus-containing supernatant was harvested, passed through a 0.45-µm-pore-size filter (Millipore), and stored at 70°C.

Fv1 transduction assay. Fv1 assays were carried out as previously described (3). Briefly, 2 × 10⁴ to 3 × 10⁴ Mus dunni cells per well were seeded on a 12-well plate. Sixteen hours later, cells were transduced with an NB-tropic delivery virus carrying a wild-type or mutant allele of Fv1 in the Fv1-IRES-EGFP cassette. Approximately 56 h later, cells were split 1:12, and after a further 16 h, they were transduced with an aliquot of N-, B-, or NB-tropic tester virus encoding EYFP which yielded 35% yellow cells in control (Fv1-null) cells. Forty-eight hours after the second transduction, cells were harvested, fixed in PBS-3.5% formaldehyde, and examined for EGFP and EYFP expression by FACS analysis with a FACS Vantage apparatus (Becton Dickinson). The XF500/T filter set (Glen Spectra) was used to separate the EGFP from the EYFP fluorescence signal. The data were analyzed with the FCSPress 1.1 package (Ray Hicks, Cambridge, United Kingdom).

Western blot analysis. M. dunni cells were transduced with delivery virus carrying one wild-type or mutant allele of Fv1 in the Fv1-IRES-EGFP cassette, as described for the transduction assay (3), under conditions where 50 to 80% of the cells were infected, as judged by the number of green fluorescent cells 2 days after infection. Approximately 2 × 10⁶ cells were lysed in 200 µl of lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate) containing complete mini-protease inhibitor cocktail (Roche). Insoluble material was removed from the samples by centrifugation. A 100-µg portion of total protein was diluted in SDS loading buffer containing 10 mM dithiothreitol and boiled for 3 min. Samples were subjected to electrophoresis on a 12% polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore) with a semidry electrotransfer apparatus (Ancos). Filters were blocked with 5% nonfat dry milk in PBS-0.1% Tween 20 and incubated for 1 h at room temperature with anti-Fv1 diluted (3:1,000) in 5% milk-PBS-0.1% Tween 20. Rabbit anti-Fv1 antiserum was prepared following repeated immunization with the gel-purified product of Fv1 expressed in Escherichia coli. After three washes in 0.5% milk-0.1% Tween 20-PBS, the filters were incubated for 1 h at room temperature in 1:1,000 solution of horseradish peroxidase-protein A (Bio-Rad) in 5% milk-PBS-0.1% Tween 20. After three washes, bound peroxidase activity was revealed with the ECL kit (Amersham Life Science) and exposed on Kodak MXB Film.

	RESULTS

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Fv1 restriction shows unique specificity for the incoming virus (3), a specificity that must result from the three differences between the predicted products of the n and b alleles of Fv1, at amino acids 358 and 399 and at the C-terminus (Fig. 1). To investigate which regions of Fv1 are necessary for restriction and what factors determine the specificity of the interaction, we have carried out a detailed genetic analysis of a series of Fv1 derivatives, measuring their ability to restrict incoming MLV.

View larger version (6K): [in this window] [in a new window]   FIG. 1.   The differences between Fv1n and Fv1b proteins. The lines are to scale and represent the Fv1 gene product. The three positions that differ between Fv1n and Fv1b, residues 358 and 399 and the C terminus, are highlighted, with the amino acids in each case indicated by their single-letter codes. Sequences for Fv1n and Fv1b can be found under GenBank accession no. X97720 and no. X97719, respectively.

C-terminal deletions. The most striking difference between the n and b alleles of Fv1 lies at the predicted C terminus. Fv1^b encodes a protein 459 amino acids long, 19 amino acids longer than the Fv1ⁿ gene product. To test the effect of these extra residues, we first deleted nucleotides in Fv1^b encoding amino acids 441 to 459, thereby truncating Fv1^b to the length of Fv1ⁿ. This mutant showed a marked increase in activity in the transduction assay against both B- and NB-tropic MLV, compared to full-length Fv1^b (Fig. 2, compare panels C and B). This implies that the long C terminus of Fv1^b is a negative factor for restriction. Next, Fv1^b and Fv1ⁿ were both deleted to produce proteins terminating at residue 437. This is the last amino acid before the C terminus that is identical in both alleles. Thus, these constructs differed only at positions 358 and 399. Fv1ⁿ terminating at residue 437 had activity identical to that of wild-type Fv1ⁿ, i.e., showing full restriction of B-tropic MLV and no restriction of N- or NB-tropic MLV (Fig. 3). Fv1^b truncated to residue 437 had the same activity as Fv1^b terminating at residue 440, fully restricting N- and NB-MLV and showing slightly less restriction of B-MLV. Interestingly, when the three amino acids that form the C terminus of the n allele (TKL) were added to the Fv1^b mutant protein that was truncated to residue 437, the previously slightly reduced restriction of B-MLV was increased to full restriction. From this it seems that restriction of B-tropic MLV is more sensitive to the specific amino acids found at the C terminus. Deleting more residues from the C terminus of Fv1, creating proteins 410 amino acids long, completely abolished restriction activity in both alleles. The differences in the restriction patterns for Fv1ⁿ and Fv1^b truncated to residue 437 indicate that the amino acids at residues 358 and 399 are important and sufficient to determine specificity and that full activity can be maintained when the C terminus is deleted.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Fv1 activity of C-terminal deletion mutants. FACS profiles illustrating the effect of the C terminus on restriction of B-tropic tester virus are shown. EGFP expression is shown on the x axis; EYFP expression is shown on the y axis. The percentages given each indicate the proportion of EYFP-positive, i.e., B-MLV-infected, cells in the EGFP-negative (Fv1) and the EGFP-positive (Fv1+) subpopulation. Wild-type Fv1n (A), mutant Fv1b441-459 (B), wild-type Fv1b (C), and mutant Fv1b411-459 (D) were introduced. The amount of restriction decreased from the full restriction shown in panel A to the partial restriction shown in panel B the slight restriction shown in panel C, and the lack of restriction shown in panel D. The percentages given are typical of the values for each level of restriction.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Summary of restriction by Fv1 C-terminal deletion mutants. The activity of each mutant against N-, B-, or NB-tropic tester virus is shown. The mutant length indicates at which residue the mutant terminates. The data for wild-type alleles are shaded. The extent of restriction is described by symbols, as follows: +, full restriction (equivalent to a reduction from 35% infection of Fv1 cells to 2% infection of Fv1+ cells, as shown by Fv1n restriction of B-MLV and Fv1b restriction of N-tropic MLV); (+), partial restriction; (), slight restriction; , no restriction.

N-terminal deletions. A series of N-terminal deletions were made to obtain five different proteins with variations in the C-terminal end: full-length Fv1ⁿ (wild-type Fv1ⁿ), Fv1ⁿ truncated to residue 437 (Fv1ⁿ438-440), full-length Fv1^b (wild-type Fv1^b), Fv1^b truncated to residue 437 (Fv1^b438-459), and Fv1^b terminating at residue 437 with the TKL sequence then added (Fv1^b438-459+TKL). Using PCR, constructs were synthesized that encoded proteins that began progressively further from the wild-type N terminus, at residues 33, 51, 62, or 93. The activities of these mutant proteins in the transduction assay are summarized in Fig. 4.

View larger version (49K): [in this window] [in a new window]   FIG. 4.   Restriction by Fv1 N-terminal deletion mutants. The activity of each mutant against N-, B-, or NB-tropic tester virus is shown. The data for wild-type alleles are shaded. The extent of restriction is indicated by symbols, as described in the legend to Fig. 3. ±, 50% restriction (half the percentage of Fv1+ cells are infected composed to the percentage infected in the Fv1 population).

Internal deletions. Even though only a relatively short region could be removed from either terminus of Fv1 without losing activity, we decided to create some internal deletion mutants to examine the role of the rest of the protein. For simplicity, only Fv1ⁿ mutants were synthesized. Constructs were created that encoded proteins with stretches of 10 amino acids deleted. Ten mutants were synthesized with deletions that resulted in the removal of amino acids between residues 109 and 384 (Fig. 5). Surprisingly, 6 out of the 10 mutants were fully active. The inactive mutants included those with deletions C-terminal of the MHR and the deletion of residues 109 to 118. Removing residues 123 to 132, only 5 amino acids along the protein, had no effect on restriction. In fact, none of the deletions of regions encoding amino acids between residues 123 and 250 caused loss of activity. Following these results, we decided to create a series of mutants with larger deletions in this region. In each case the construct synthesized resulted in the deletion of two sections previously deleted and the region in between them. All but the largest deletion (removal of 128 amino acids) had no effect on activity. This mutant, Int.2-7, showed only 50% restriction. However, as the regions deleted in separate mutants overlapped and covered the whole of the region encoding residues 123 to 250, it is clear that the specific residues found in this section of the protein are dispensable for activity. Further, taken together with the data from the preceding section, these results suggest the presence of two functional domains in Fv1.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Restriction of Fv1n internal deletions. The diagram at the top shows the major features of Fv1n, with the lines below representing mutants. The stretch of sequence deleted is indicated (open boxes). The name of each mutant is shown to the left of each line, and its activity against B-tropic MLV is shown to the right. Symbols indicate the extent of restriction, as follows: +, complete; ±, 50% restriction , none.

MHR mutants. Capsid proteins from all known replication competent viruses except spumaviruses contain an MHR (25). The function of this region is unclear, but mutation of the conserved residues causes several defects in virus replication, including blocking of assembly and release of viral particles, and production of noninfectious virions (5, 17, 25). Despite not requiring budding, maturation, or infection functions for activity, sequence comparisons with capsid have identified an MHR in Fv1 (1). This may be a remnant of Fv1's evolutionary past and have no function, or it may serve some purpose in restriction. To determine the importance of this motif for Fv1 activity, each of the invariant residues in the MHR was mutated by site-directed mutagenesis to amino acids that had caused an effect in HIV (17) or RSV (5) capsid proteins, and the activity of each mutant was measured in the transduction assay. The results are shown in Fig. 6.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Restriction of Fv1 MHR mutants. The diagram shows the MHR sequence of wild-type Fv1 with the single-amino-acid changes made shown below. The consensus sequence is written at the bottom ( and O represent aromatic and aliphatic amino acids, respectively). The extent of restriction of Fv1n and Fv1b with each mutation is shown on the right. +, restriction; , no restriction; n/d, not determined.

Protein expression. To ensure that the loss of activity seen in certain mutants was due to the effects of the specific mutation and not to a defect in protein expression or stability, Western blot analysis was carried out on cell lysates of M. dunni cells expressing each mutant. Figure 7 shows representative examples of each of the four types of mutants referred to above. For every mutant analyzed, including all the C-terminal deletion mutants, all the N-terminal deletions of Fv1ⁿ and Fv1ⁿ438-440, several N-terminal deletions of Fv1^b derivatives, all the inactive internal deletion mutants, A271P and V282E MHR mutants, and all the alanine specificity mutants, a protein of the correct size was identified by anti-Fv1 antibodies (Fig. 7 and data not shown). This indicates that each mutant was synthesized in M. dunni cells. We have been unable to detect wild-type Fv1 in N-3T3 or B-3T3 cells by Western blot analysis (data not shown), confirming the reported low-level expression in these cell lines (2). Thus, Fv1 is expressed at higher levels from our constructs than natural Fv1. However, even with these elevated expression levels, some of these mutant proteins were unable to restrict incoming MLV, implying that the region mutated is necessary for activity.

View larger version (79K): [in this window] [in a new window]   FIG. 7.   Detection of mutant Fv1 proteins. Shown are the results of Western blot analysis of M. dunni cells transduced with Fv1 derivatives Lanes: a, negative control; b, wild-type Fv1n; c, wild-type Fv1b; d, C-terminal deletion mutant Fv1n1-410; e, C-terminal deletion mutant Fv1b1-410; f, N-terminal deletion mutant Fv1n1-201; g, internal deletion mutant Int.3-6; h, MHR mutant Fv1nE273D. Virus input was adjusted to result in 50 to 80% transduction levels as measured by EGFP expression. Total protein (100µg) was loaded in each lane, and Fv1 was detected with anti-Fv1 polyclonal antibody. Wild-type Fv1 and mutant Int. 3-6 are active; the remaining mutants are inactive.

Specificity mutants. To examine the relative roles of the three differences between Fv1ⁿ and Fv1^b, we had already constructed a series of mix-and-match derivatives (3). Results from these mutants as well as the Fv1ⁿ and Fv1^b proteins, with and without their normal C termini, are summarized in Fig. 8A. Herein, Fv1 derivatives are described in three-letter codes depending on whether they have n or b sequences at positions 358, 399, and the C terminus (thus, e.g., the derivative nbn has the n allele sequence at 359 (Lys), the b allele Arg at 399 and the TKL of n origin at the C terminus) (Fig. 8).

View larger version (39K): [in this window] [in a new window]   FIG. 8.   Restriction of specificity mutants. Mutants are named with three letters; the first refers to position 358, the second to position 399, and the third to the C-terminus of the allele. n indicates the mutant is Fv1n-like at that position, b indicates it is Fv1b like at that position, A means that the residue has been mutated to alanine, and a dash in the third position indicates that the allele terminates at residue 437. Symbols are used to indicate the extent of restriction, as follows: +, full restriction; (+), partial restriction; (), slight restriction; , no restriction.

	DISCUSSION

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The mechanism of Fv1 restriction is unknown but is thought to involve a specific interaction between the MLV CA protein and the Fv1 gene product (3, 7). To probe the regions of Fv1 that are important for activity, we prepared a series of Fv1 mutants and examined their ability to restrict virus replication. These studies permit a number of conclusions. First, sequences toward the N and C termini of Fv1 are necessary for function, but perhaps surprisingly, one-third of the specific internal sequences are dispensible (amino acids 123 to 250). Second, specific sequences within the MHR are essential for function. Third, restriction specificity appears to be a property of a combination of amino acids located within the C-terminal portion of Fv1. Finally, the fact that such a wide range of mutations affect Fv1 function seems to rule out the possibility that Fv1 is acting as RNA (29); instead, they provide evidence for a direct interaction between CA and the Fv1 gene product.

If the C-terminal third of the protein is responsible for the specificity of Fv1 binding, what role does the N-terminal region play? One possibility is that the N- and C-terminal domains interact to form one binding pocket. This is consistent with the observation that the nature of the C terminus affects the function of N-terminal deletions (Fig. 4). One of the amino acids deleted in the inactive mutant Int.1 is a Cys which might form a disulphide bond with a C-terminal Cys (370 or 411). However, this is unlikely in a cytoplasmic protein, and site-directed mutagenesis of the Cys to an Ala does not affect restriction (data not shown). Alternatively, the N-terminal domain might play some other role essential for Fv1 function, such as providing a cellular localization signal.

The MHR region is highly conserved within retroviruses (1) but its functional significance remains to be fully elaborated (26). Fv1 also contains the same motif, with identical mutations preventing virus assembly or maturation and inhibiting Fv1 function, apparently lending further weight to the idea that the viral origin of Fv1 is important for function (7). However, attempts to align Fv1 and CA, using a sophisticated threading program (27), have not revealed any further similarities between the two proteins (W. Taylor, personal communication). Structural studies of a variety of CA proteins (4, 10, 12) indicate that much of the MHR forms a linker region joining separate domains of CA; our finding of two functional domains in Fv1 is consistent with a similar role for the MHR in Fv1. Resolution of this issue must await structural studies on Fv1.

When the sequences of the n and b alleles of Fv1 were first determined (2), a striking inverse relationship between the charge of amino acid 358 on Fv1 and that of amino acid 110 on restricted virus was noted, suggesting the possibility that a salt bridge might be important for restriction. This appears not to be the case. Our results with the mix-and-match mutants and the Ala substitution at 358, as well as the observation that MLV with a Trp at CA position 110 is N-tropic (13), are inconsistent with this notion. Rather, the introduction of a specific charged residue in the viral capsid will prevent restriction. This suggests a complex evolutionary interplay between virus and Fv1, with viral changes to allow replication selecting different Fv1 variants capable of restricting virus. These changes to CA can occur in multiple positions; we have preliminary evidence that alterations in at least 6 amino acids of CA can influence restriction by Fv1 (A. Stevens and M. Bock, unpublished data) and that minor alleles of Fv1 have other changes (S. Ellis and J. P. Stoye, unpublished data). It appears likely that the Fv1-CA interaction is complex, with multiple amino acids on both sides playing important roles. Again, structural information appears essential for understanding the interaction in detail.