Abrogation of Fv-1 Restriction by Genome-Deficient Virions Produced by a Retrovirus Packaging Cell Line

Transcription

1 JOURNAL OF VIROLOGY, JUlY 199, p X/9/ $2./ Copyright 199, American Society for Microbiology Vol. 64, No. 7 Abrogation of Fv-1 Restriction by Genome-Deficient Virions Produced by a Retrovirus Packaging Cell Line LAWRENCE R. BOONE,t* CYNTHIA L. INNES, AND CATHERINE KREBS HEITMAN Cellular and Genetic Toxicology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 2779 Received 5 December 1989/Accepted 29 March 199 The Fv_lb-mediated restriction of N-tropic retrovirus vector infection of BALB/3T3 cells was partially abrogated by prior infection with N-tropic murine leukemia virus. Likewise, abrogation of the FV_lb restriction of N-tropic murine leukemia virus replication was accomplished by prior infection with genome-deficient virions produced by an N-tropic murine leukemia virus packaging cell line. The latter observation suggests that the Fv-1 target in genome-deficient virions abrogates Fv-1 restriction in the absence of any viral genomedirected processes. The mouse Fv-1 gene governs the relative sensitivity of cells to infection by certain host range classes of murine leukemia viruses (MuLV) (14, 24, 26). The target for Fv-J restriction is the 3-kilodalton capsid protein known as CA or p3 (28), and differences in two adjacent amino acids in CA determine whether a virus is sensitive to the product of the n or b allele of Fv-J (7, 23). A virus restricted by the b allele is not restricted by the n allele and is therefore designated N-tropic. Conversely, a virus restricted by the n allele is not restricted by the b allele and is designated B-tropic. Restriction is not absolute and is generally observed as a 1.5- to 3-log reduction in infectivity relative to permissive cells. The titration of infectivity in restrictive cells is frequently two-hit (or greater), suggesting that more than one infectious unit is required to overcome Fv-1 restriction and result in a successful infection (6, 8, 22, 25, 32, 33). It has been proposed that the first virus to infect a restrictive cell is blocked by Fv-J and does not replicate, but initiates a biochemical process that results in the transient abrogation of Fv-1 (1, 8). A subsequently encountered virus is therefore able to replicate without interference. The abrogating virus must be of the restricted tropism, but does not have to be able to replicate (e.g., it may be heat inactivated) (1, 2). Although the multihit requirement for infectivity has been extensively documented, some investigators have reported single-hit infectivity in restrictive cells (16, 29). We have recently described the development of an N-tropic (i.e., Fv-lb_sensitive) packaging cell line and have observed restriction (approximately 1-fold) in Fv-lb cells infected with a retrovirus vector produced by it (4). The titration pattern was always single-hit, in contrast to our experience measuring infectivity of N-tropic MuLV in restrictive cells using the XC plaque assay in restrictive cells. Although single-hit infectivity suggests that abrogation is not essential to infect some cells in a restrictive culture, we present evidence in this report that specific abrogation of Fv-J restriction could be accomplished that allowed additional cells to become infected with the vector. Furthermore, the genome-deficient * Corresponding author. t Permanent address: Division of Virology, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, NC virions produced by the N-tropic packaging cell sufficient to abrogate Fv-lb restriction. MATERIALS AND METHODS line were Cells and viruses. Mink lung CCL 64 cells (15) and the NB-tropic (also amphotropic) retrovirus packaging cell line PA317 (2) were obtained from the American Type Culture Collection. The NB-tropic packaging cell lines *CRE (ecotropic) and ticrip (amphotropic) were generously provided by R. Mulligan. The Fv-J N-tropic retrovirus packaging mutant provirus was derived from pwn41 (5), a molecular clone of WN182N (14, 22). The resulting packaging cell line, N-Pac, and the retrovirus vector conferring G418 resistance, RV-neo (provided by E. Linney), have been described previously (4). B-tropic MuLV was derived from pwb5 (5), a molecular clone of WN182B (14, 22). A chimeric N-tropic MuLV (designated pwb/n69) was derived from a clone of pwb5 containing the Fv-J tropism target from pwn41. Similarly, a chimeric Nr_tropic MuLV (designated pwb/gn2) was derived from pwb5 containing the Fv-J tropism target of Gross MuLV clone pg14. N' tropism refers to the subtype of N tropism that is not restricted by the Fv-1nr allele (Hartley and Rowe, cited in 19 and 31). The murine cell lines BALB/3T3 and SC-1 and rat XC cells were originally obtained from R. W. Tennant, National Institute of Environmental Health Sciences, Research Triangle Park, N.C. CCL 64, PA317, and N-Pac cells were maintained in Dulbecco modified Eagle medium (GIBCO Laboratories) supplemented with 8% fetal bovine serum (GIBCO), 1 U of penicillin, and.1 mg of streptomycin per ml. XC, BALB/3T3, and SC-1 cells were maintained in modified Eagle medium (GIBCO) supplemented with 4% fetal bovine serum and antibiotics as above. qcrip and *CRE were maintained in Dulbecco modified Eagle medium supplemented with 1% defined/supplemented calf serum (Hyclone Laboratories, Inc.). Virus or packaged vector stocks (24-h harvests) were produced in the described medium for the particular cell line supplemented with 1-6 M hydrocortisone and.1 U of insulin per ml, filtered through a membrane (pore size,.45 ixm), and stored as aliquots over liquid nitrogen. N-tropic RV-neo vector stocks produced in N-Pac cells are desig- Downloaded from on November 15, 218 by guest

2 VOL. 64, 199 nated RV-neo (N-Pac), and NB-tropic stocks produced in PA317 cells are designated RV-neo (PA317). Abrogation protocol. Hydrocortisone- and insulin-supplemented media (as above) from packaging cell lines (N-Pac, PA317, tjcrip, and *4CRE) were collected, filtered through a.45-pum membrane, and stored frozen over liquid nitrogen. Abrogation was usually performed by exposing cells to replication-competent virus stocks (multiplicity of infection, 3 XC PFU per cell) or packaging cell line medium (undiluted) in the presence of 16,ug of Polybrene per ml for 2 h. At the specified time, usually 6 h beyond the initial exposure to abrogating virus, cells were infected with the challenge virus to be assayed. Standard protocol for virus infection and assay. Helper virus-free packaged vector or replication-competent virus infections were routinely performed 1 day after plating by treating cell cultures (in 6-mm-diameter dishes or 35-mm six-well plates) with 1 ml of virus dilution in the presence of 16,ug of Polybrene. The inoculum was usually removed after 2 h, and cultures were refed with routine maintenance medium and maintained for G418-resistant colony assay or XC plaque assay (27). For G418-resistant colony formation, culture medium was supplemented with 5,ug of G418 per ml beginning the day after infection (i.e., day 3). During antibiotic selection, cultures were routinely refed with medium containing 1 mg of G418 per ml every 3 to 4 days and stained on day 15 for counting. Reverse transcriptase assay. Culture medium was passed through a.45-,um filter and assayed for reverse transcriptase by a modification of the protocol described by Goff et al. (12). A 2-,ul portion of medium was mixed with 4,uls of a cocktail to give final concentrations of 5 mm Tris hydrochloride (ph 8.3), 6 mm NaCl,.6 mm MnCl2, 2 mm dithiothreitol,.5% Nonidet P-4, 22,ug of poly(ra- dt)12_18 per ml, and.5,um [3H]dTTP (67 Ci/ mmol). The reaction was incubated at 37 C for 9 min, and a 5-g±l sample was spotted onto a 1-in. (2.54-cm) square of DE81 paper (Whatman Inc.). Unincorporated [3H]dTTP was removed by washing in 5% Na2HPO4. Filters were washed briefly in H2 and finally in 95% ethanol prior to drying and counting in Ready-Safe (Beckman Instruments, Inc.) liquid scintillation cocktail. Virion RNA analysis. Cell culture medium used in abrogation experiments was analyzed for virion RNA content by a slot blot hybridization protocol. Filtered medium (.45-,um filter) was centrifuged at 47, rpm for 1 h in a TLS55 rotor in a Beckman TL1 ultracentrifuge. The pellet was suspended in 1 mm Tris hydrochloride (ph 7.5)-l mm EDTA-.5% sodium dodecyl sulfate at 65C to O.1x the original volume. Then 5,ul of the sample or 5 RI of serial 1:5 dilutions was adjusted to a final volume of 2,u containing ABROGATION OF Fv-1 RESTRICTION % formamide, 6% formaldehyde, and 1 mm sodium phosphate and heated at 65 C for 15 min. An equal volume of 2 x SSC (lx SSC is.15 M NaCl plus.15 M sodium citrate) was added, and RNA was collected by ifitration onto a nitrocellulose membrane by using a minifold II slot blot apparatus (Schleicher and Schuell, Inc.). Samples were washed by additional filtration with lox SSC, and the membrane was dried at 8 C for 2 h in a vacuum oven. Viral RNA was detected by hybridization with a 32P-labeled DNA probe. A molecular clone of WN182N MuLV, pc18, was 32p labeled by a random-primer protocol (Bethesda Research Laboratories, Inc.) and hybridized to the membrane-bound RNA for 65 h at 65 C in 5 x SSC-5 x Denhardt solution-2 mm Tris hydrochloride (ph 7.5)-.1% sodium dodecyl sulfate-1p,g of sheared calf thymus DNA per,ul. Posthybridization washing was carried out in 2x SSC-1.% sodium dodecyl sulfate twice briefly at room temperature and for 3 min at 65 C. This was followed by an additional wash in 2x SSC-.1% sodium dodecyl sulfate for 3 min at 65 C and twice briefly at room temperature. A high-stringency wash was omitted to detect possible packaging of MuLV-related sequences in the genome-deficient virions. The dried membrane was exposed with an intensifier screen (Du Pont) to Kodak XAR5 X-ray film at -7 C. Exposure revealed faint bands at -7 C for 6 days. Reagents. Polybrene (hexadimethrine bromide) was purchased from Aldrich Chemical Co., Inc., and G418 was purchased from GIBCO. RESULTS Abrogation of Fv-1 restriction by replication-competent MuLV. Recent results indicated that BALB/3T3 cells (Fv-lb) were approximately 1-fold less sensitive to infection by an N-tropic packaged vector than were AKR cells (Fv-J') (4). The infectivity titration pattern was single-hit in both permissive and restrictive cells. Although a single-hit pattern in restrictive cells suggests that abrogation is not essential for infection of some cells of the restrictive genotype, our results did not exclude the possibility that abrogation occurs under some circumstances. If abrogation could be accomplished in this system, it would be expected to increase the observed G418-resistant colony titer by making more BALB/ 3T3 cells temporarily susceptible to infection. To test for abrogation, we preinfected BALB/3T3 cells with N-tropic (i.e., restricted) or B-tropic (i.e., nonrestricted) MuLV 6 h prior to challenge with the antibiotic-selectable vector RVneo. This vector was packaged either by the N-tropic packaging cell line N-Pac or the NB-tropic packaging cell line PA317. The data shown in Fig. 1 indicate that preinfection with either N-tropic or Nr-tropic MuLV isolates (both are sensitive to Fv-1b) increased the infectivity of RVneo(N-Pac) relative to mock-preinfected controls. Preinfection with B-tropic MuLV did not. Thus, Fv-lb restriction was disarmed or abrogated, to some extent, by exposure to restricted virus but not by exposure to virus of the homologous tropism. Furthermore, challenge infection by RVneo(PA317) was relatively unaffected by prior infection with all viruses tested. These observations are consistent with the published specificity offv-j abrogation (3, 8, 21, 33). Abrogation of Fv-1 restriction by genome-deficient MuLV. The previous experiment demonstrated that replicationcompetent N-tropic MuLV could abrogate the Fv-1b_mediated restriction of RV-neo(N-Pac) infection. It has been shown that the ability of a virus to replicate is not required for it to abrogate (1). In that regard, the genome-deficient (replication-defective) virions produced by the packaging cell lines might be expected to be capable of abrogation. Although the ability to replicate is not required for abrogation, the model proposed by Bassin et al. (1, 2) implicated virion RNA in the abrogation mechanism. According to that model, virions lacking MuLV-specific coding sequences would be unable to abrogate. Thus, the genome-deficient virions produced by the packaging cell line might not be capable of abrogating Fv-1 restriction. The packaging mutant we have constructed (4) allows us to directly test that hypothesis by exposing Fv-lb cells to the genome-deficient N-tropic virions (in the absence of a packageable vector) and then challenging the Fv-l restriction by infection with N- tropic MuLV. The genome-deficient virions are not capable of producing Downloaded from on November 15, 218 by guest

3 3378 BOONE ET AL. J. VIROL. 5r RV-neo ( N-Pac) Experiment 1-9. S 1.) re) co I- -r 4 o cj c._l ao z 4O o 4 3C 21 1o RV-neo(N-Poc) mock B N N' RV-neo(PA317) RV-neo(PA317) mock B N Experimrnt 2 FIG. 1. Pretreatment of BALB/3T3 with MuLV and subsequent challenge with RV-neo(N-Pac) or RV-neo(PA317). BALB/3T3 cells were mock treated or infected with N-, Ne-, or B-tropic MuLV (multiplicity of infection, 3) for abrogation as described in Materials and Methods. Six hours later, cells were infected with RV-neo(N- Pac) or RV-neo(PA317) at various dilutions, and G418-resistant colony formation was assayed as described in Materials and Methods. The results of two independent experiments are shown. The data are presented as the average of duplicate cultures infected with challenge virus [at a dilution which produced colonies within a countable range: for RV-neo(N-Pac), the dilution shown is 3.2 x io- for both experiments; for Rv-neo(PA317), the dilution is 1 in experiment 1 and 1-4 in experiment 2]. Error bars indicate the range of values. XC plaques and have no infectious activity except for the ability to package retrovirus vectors (4). To have some measure of virion concentration, we performed reverse transcriptase assays on medium from the control and various packaging cell lines. The data presented in Fig. 2 demonstrate that reverse transcriptase is detectable in all packaging cell line samples, although at lower levels than in medium from replication-competent virus. No attempt was made to normalize samples on the basis of reverse transcriptase activity; rather, undiluted culture medium was used to achieve the maximnum degree of abrogation or an appropriately rigorous control. In addition, to directly demonstrate that the packaging cell-produced virions are in fact deficient in genomic RNA, we performed slot blot analysis of pelleted particles from cell-free medium as described in Materials and Methods. The largest amount of each sample is equivalent to 5,ul of culture medium followed by 1:5 serial dilutions. The autoradiograph is shown in Fig. 2. It is obvious that medium from cells producing replication-competent virus hybridize strongly to the MuLV-specific probe and can be faintly detected at the lowest level tested, corresponding to.16,ul of culture medium. Only faint hybridization is detected in the PA317 and IJCRE samples, and none is detected in the icrip, N-Pac, or CCL 64 samples. Although some small amount of RNA may be packaged by PA317 and 2 r CL c E9 s k r- IL x v) co z-z S_- j _~ ~ ~ E5 ; 5 E~ r.16 Co * z FIG. 2. Reverse transcriptase activity and genomic RNA content of virions. (Top) Reverse transcriptase activity was determined as described in Materials and Methods. (Bottom) Hybridization analysis of cell-free medium was performed as described in Materials and Methods. Numbers refer to the volume of original culture medium each sample represents (in microliters). The autoradiograph is overexposed for some samples to reveal faint bands in others. qjcre, all of the packaging cell-produced virions are clearly genome deficient. The B-tropic virus-producing culture and PA317 have roughly the same reverse transcriptase activity, yet differ by more than 1-fold in the presence of virion RNA detectable by hybridization. BALB/3T3 cells were exposed to undiluted culture medium from the various cell lines and challenged 6 h later by infection with N-tropic MuLV. Infectivity of the challenge virus was assayed by XC plaque formation. Medium from the CCL 64 cell line, from which N-Pac was developed, was sed as a control. The data in Fig. 3 demonstrate that preinfection of BALB/3T3 cells with medium from the N-tropic packaging cell line increased the XC plaqueforming ability of N-tropic MuLV (i.e., abrogated Fv-Jb restriction), whereas the use of control cell medium did not. Medium from the NB-tropic packaging cell lines PA317, tpcrip, and *CRE also did not increase the challenge virus infectivity, consistent with a requirement for virus of the restricted phenotype to abrogate Fv-J restriction. The reverse transcriptase activity in medium from these NB-tropic packaging cell lines is equal to or higher than in medium from N-Pac (Fig. 2), thus providing an appropriate comparison. Lower dilutions of N-tropic MuLV resulted in too many XC plaques to count in the N-Pac-pretreated cells, and higher dilutions resulted in too few XC plaques to count in the CCL 64-pretreated cells. Challenge with B-tropic virus was also performed following preinfection with N-Pac or control CCL 64 medium. The data in Fig. 3 demonstrate that the N-Pac pretreatment had no effect on the permissive combination of Fv-lb cells with B-tropic virus infection. Abrogating activity was greatly diminished when dilutions of N-Pac medium (e.g., 1:1) were tested (data not shown). This suggested that the number of virion Fv-J target molecules present in undiluted medium is not in vast excess with respect to available Fv-1 gene product in BALB/3T3 cells. To find whether multiple doses of undiluted medium could improve the degree of abrogation, a single dose given 8 h prior to MuLV challenge infection was compared with a Downloaded from on November 15, 218 by guest

4 VOL. 64, 199 ABROGATION OF Fv-1 RESTRICTION p N-Tropic MuLV B -Tropic MuLV a 1 UL. C.) I- I'5 1-J m to 4 co -j. c U z 2F 1c A' V 1- a;, a FIG. 3. Pretreatment of BALB/3T3 cells with medium from various packaging cell lines. BALB/3T3 cells were treated with undiluted medium from CCL 64, N-Pac, PA317, *CRIP, and qjcre cells for the abrogation assay as described in Materials and Methods. N-tropic (at a 1.25 x 1o-3 dilution) and B-tropic (at a 5 x lo-3 dilution) MuLV were used to challenge these cells 6 h later. The challenge was followed by a standard XC plaque assay. The average plaque counts of triplicate (for N-Pac and CCL 64) or duplicate (for *CRIP, ipcre, and PA317) dishes are shown, with bars marking the range. triple dose given at 8, 6, and 3 h prior to challenge infection (Fig. 4). The single-dose procedure was also used with permissive SC-1 cells (13). As expected, N-tropic MuLV had a higher titer in SC-1 cells than in BALB/3T3 cells, and prior exposure to N-Pac medium did not further increase the infectivity in SC-1 cells. Consistent with the data in Fig. 3, a single exposure of BALB/3T3 cells to N-Pac medium increased the titer of N-tropic MuLV. The multiple-exposure protocol increased the degree of abrogation over that of a single dose and brought N-tropic MuLV infectivity almost to the level observed in the permissive SC-1 cell culture. The effectiveness of the N-Pac medium in abrogating Fv-1 restriction is dependent upon the timing of treatment relative to virus challenge. In our study, a window of 3 to 8 h before challenge was optimal in abrogating Fv-J restriction; however, some abrogating activity was observed with treatments 24 h prior to the challenge and 3 h after the challenge (data not shown). These results are consistent with the observations of others concerning abrogation with replication-competent (8) and inactivated (1, 3) virions. DISCUSSION The single-hit infectivity pattern previously observed in RV-neo(N-Pac) infection of BALB/3T3 cells suggested that some genotypically restrictive cells are phenotypically permissive without a requirement for a first hit of restricted virus (4). Perhaps in some cells the level of the Fv-1 gene product is below a threshold necessary for restriction and therefore abrogation is not required. In the experiments presented here, it is clear that specific abrogation was accomplished by exposure of cells to restricted MuLV. Additional restrictive cells therefore became successfully (o' l 1 I ml I- ax -J ro 4Vf Co En ~ X a- C-) VIRUS DILUTION FIG. 4. Comparison of single and multiple pretreatments of abrogating medium. BALB/3T3 cells ( ) were exposed to undiluted medium from N-Pac cells or control CCL 64 cells either at 8 h preinfection or for a total of three treatments at 8, 6, and 3 h prior to infection with N-tropic MuLV. SC-1 cells ----) were pretreated at the single time point only. Dilutions of N-tropic MuLV were used to challenge the pretreated cells. The average XC plaque count of triplicate dishes was plotted against the virus dilution. Error bars indicate standard deviation. Where datum points are tightly clustered on the graph, only the extreme deviations are drawn in. Symbols (single exposure): *, N-Pac;, CCL 64; O, mock infected. Symbols (multiple exposure): A, N-Pac; A, CCL 64;, mock infected. infected with the antibiotic-selectable vectors. It is obvious in Fig. 1 that there is a difference between the abrogating ability of the N-tropic and Nr_tropic virus stocks, even though they are both adjusted to a multiplicity of infection of 3. Although this may reflect a true biological difference in these two viruses, there are so many poorly understood variables at play in Fv-1 restriction and abrogation that we do not wish to draw any firm conclusions from this difference in magnitude. The most impressive and consistent feature of Fv-1 abrogation is the genetic specificity rather than the degree of abrogation seen from one experiment to another. The experiments whose results are shown in Fig. 1 and 3 also demonstrate different magnitudes of abrogation. The precise reason for this discrepancy is unknown, but may be related to the difference in assay systems (G418 resistance versus XC plaque) as well as the differences in abrogating virus (MuLV versus N-Pac). Most importantly, our results indicate that genome-deficient virions produced by N-tropic packaging cell lines were sufficient to abrogate Fv-Jb restriction. Although the ability to make valid comparisons is quite limited, the genomedeficient virions do not appear to be less active in abrogating capacity than are the replication-competent viruses. Our interpretation of these findings is that an appropriate presentation of the Fv-J target on the CA protein of newly penetrated virions is sufficient to abrogate the restriction. We suggest that this occurs by specific binding to and Downloaded from on November 15, 218 by guest

5 338 BOONE ET AL. titrating out the available Fv-J gene product. A specific role for virion RNA is apparently not required. This is in contrast to the model proposed by Bassin et al. (1, 2). That model was based on several observations, including the finding that abrogation did not require reverse transcription, and loss of abrogating activity at 43 C was correlated with loss of intact 35S RNA.- However, the ability of core particles to bind the Fv-1 gene product may require a structural integrity that parallels the stability of genomic RNA. Furthermore, the ability of virions to attach to and penetrate cells, steps necessary for abrogation, may coincidentally parallel the stability of genomic RNA. Thus, some of the early published data do not discriminate between the model we propose and that of Bassin et al. An observation that strongly supported the model of Bassin et al. was the finding that genomedeficient virions produced by cells treated with dactinomycin (11, 17, 18) did not abrogate Fv-1 restriction (2). On the basis of that experiment, the presence of virion targets in newly infected cells was insufficient to abrogate, thus leading to the conclusion that genomic RNA was required. Together with their observations that reverse transcription was not required for abrogation, these workers concluded that the RNA served some function other than as template for viral DNA synthesis. A suggested possibility was that this RNA served as early mrna, a concept based on several other reports (1, 3). We cannot explain why virions produced by cells treated with dactinomycin do not abrogate Fv-J restriction whereas N-Pac virions do. Since the mechanism of genome deficiency in virions produced by cells treated with dactinomycin is not clearly established, it is possible that the particles are structurally aberrant and do not properly present the Fv-J target. The particles produced by cells transfected with the packaging-deficient provirus may more closely resemble authentic virions. It has been previously shown that cells chronically infected with N-tropic virus remain restrictive (9). Taken together with the results presented here, we suggest that de novo synthesized Fv-l target, in the form of pr659ar, is not accessible to the Fv-J gene product. Differential compartmentalization of outgoing virus particles or protection of the Fv-1 target epitope in precursor molecules due to folding may be responsible for this apparent lack of interaction. The experiments reported here suggest that abrogation occurs when the Fv-1 gene product is titrated below the threshold necessary for restriction. The controversy over single-hit and two-hit infectivity titration patterns in Fv-J restriction may be related to uncontrolled variables in individual experimental systems. For example, both the percentage of cells in which the Fv-1 product is below the threshold for restriction and the concentration of particles capable of abrogating but not replicating could affect'the titration pattern. When the probability that a single infectious virus particle infects a cell with a subthreshold level of Fv-J (i.e., phenotypically permissive) is greater than the probability that it infects a cell abrogated by another virus particle (replication competent or not), the pattern will shift from two-hit to single-hit. Alternatively, there may simply be a stochastic process whereby the virus occasionally escapes the restriction, independent of the level of the gene product. Consistent with either hypothesis, single-hit tails at high virus dilution have been found in predominantly two-hit systems (25). In our experience with the XC plaque assay, multihit patterns are frequently observed in restrictive cells, in contrast to our observations with the G418-resistant colony assay. The precise pattern (i.e., two-hit or greater) J. VIROL. has been difficult to determine and is not always consistent. Experimental abrogation has previously been shown to shift a two-hit pattern to a single-hit pattern (8). In experiments in which we clearly observe a multihit XC plaque infectivity in restrictive cells, experimental abrogation with N-Pac virions shifts the pattern to single-hit (L. Boone and C. L. Innes, unpublished data). Perhaps the reason we do not see multihit kinetics in BALB/3T3 cells infected with Rv-neo(N-Pac) (4) is that the dilutions used to assay infectivity are beyond the range in which abrogating activity could be detected. A complete understanding of the mechanism of Fv-1 restriction will require identification of the Fv-J gene product. The Fv-J system is attractive for further study because of the potential for modeling antiviral therapy targeted to the capsid protein of human immunodeficiency virus and other human retroviruses. ACKNOWLEDGMENTS We thank R. W. Tennant and W. K. Yang for helpful discussion and encouragement in these studies. We appreciate the help of S. Stasiewicz and K. Cowardin in preparing the figures and manuscript and thank Katyna Borroto-Esoda for setting up the virion RNA slot blot analysis. We also thank K. R. Tindall and J. Stowers for critical review of the manuscript. LITERATURE CITED 1. Bassin, R. H., G. Duran-Troise, B. I. Gerwin, and A. Rein Abrogation of Fv-1b restriction with murine leukemia viruses inactivated by heat or by gamma irradiation. J. Virol. 26: Bassin, R. H., B. I. Gerwin, J. G. Levine, G. Duran-Troise, B. M. Benjers, and A. Rein Macromolecular requirements for abrogation of Fv-1 restriction by murine leukemia viruses. J. Virol. 35: Benjers, B. M., R. H. Bassin, A. Rein, B. I. Gerwin, and G. Duran-Troise Mechanism of restriction of murine leukemia viruses varies between different strains of Fv-1n mice. Int. J. Cancer 24: Boone, L. R., C. L. Innes, P. L. Glover, and E. Linney Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics observed in restrictive cells. J. Virol. 63: Boone, L. R., F. E. Myer, D. M. Yang, C.-Y. Ou, C. K. Koh, L. E. Roberson, R. W. Tennant, and W. K. Yang Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes. J. Virol. 48: Decleve, A.,. Niwa, E. Gelmann, and H. S. Kaplan Replication kinetics of N- and B-tropic murine leukemia viruses on permissive and nonpermissive cells in vitro. Virology 65: DesGroseillers, L., and P. Jolicoeur Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses. J. Virol. 48: Duran-Troise, G., R. H. Bassin, A. Rein, and B. I. Gerwin Loss of Fv-1 restriction in Balb/3T3 cells following infection with a single N tropic murine leukemia virus particle. Cell 1: Duran-Troise, G., R. H. Bassin, B. F. Wallace, and A. Rein Balb/3T3 cells chronically infected with N-tropic murine leukemia virus continue to express Fv-1b restriction. Virology 112: Gallis, G. M., R. N. Eisenman, and H. Diggelman Synthesis of the precursor to avian RNA tumor virus internal structural proteins early after infection. Virology 74: Gerwin, B. I., and J. G. Levin Interactions of murine leukemia virus core components: characterization of reverse transcriptase packaged in the absence of 7S genomic RNA. J. Virol. 24: Goff, S., P. Traktman, and D. Baltimore Isolation and Downloaded from on November 15, 218 by guest

6 VOL. 64, 199 properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J. Virol. 38: Hartley, J. W., and W. P. Rowe Clonal cell lines from a feral mouse embryo which lack host-range restrictions for murine leukemia viruses. Virology 65: Hartley, J. W., W. P. Rowe, and R. J. Huebner Host range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5: Henderson, I. C., M. M. Lieber, and G. Todaro Mink cell line MvlLu (CCL 64). Focus formation and generation of "non-producer" transformed cell lines with murine and feline sarcoma viruses. Virology 6: Jolicoeur, P., and D. Baltimore Effect of the Fv-1 locus on the titration of murine leukemia viruses. J. Virol. 16: Levin, J. G., P. M. Grimley, J. M. Ramseur, and I. K. Berezesky Deficiency of 6 to 7S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. J. Virol. 14: Levin, J. G., and M. J. Rosenak Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. Proc. Natl. Acad. Sci. USA 73: Mayer, A., M. L. Duran-Reynals, and F. Lilly Regulation of lymphoma development and of thymic, ecotropic and xenotropic MuLV expression in mice of the AKR/J and RF/J cross. Cell 15: Miller, A. D., and C. Buttimore Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6: Niwa, O., A. Decleve, and H. S. Kaplan Conversion of restrictive mouse cells to permissive during sequential and mixed double infection by murine leukemia viruses. Virology 74: O'Donnell, P. V., C. J. Deitch, and T. Pincus Multiplicitydependent kinetics of murine leukemia virus infection in Fv- 1-sensitive and Fv-1-resistant cells. Virology 73: Ou, C.-Y., L. R. Boone, C.-K. Koh, R. W. Tennant, and W. K. ABROGATION OF Fv-1 RESTRICTION 3381 Yang Nucleotide sequences of gag-pol regions that determine the Fv-1 host range property of BALB/c N-tropic and B-tropic murine leukemia viruses. J. Virol. 48: Pincus, T., J. W. Hartley, and W. P. Rowe A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses. J. Exp. Med. 133: Pincus, T., J. W. Hartley, and W. P. Rowe A major genetic locus affecting resistance to infection with murine leukemia viruses. IV. Dose-response relationships in Fv-I-sensitive and resistant cell cultures. Virology 65: Pincus, T., W. P. Rowe, and F. Lilly A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major locus described for resistance to Friend murine leukemia virus. J. Exp. Med. 133: Rowe, W. P., W. E. Pugh, and J. W. Hartley Plaque assay techniques for murine leukemia viruses. Virology 42: Schindler, J., R. Hynes, and N. Hopkins Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Virol. 23: Schuh, V., M. E. Blackstein, and A. A. Axelrad Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: titration patterns in strains SIM and SIM.R congenic at the Fv-1 locus. J. Virol. 18: Shurtz, R., S. Dolev, M. Aboud, and S. Salzberg Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus. J. Virol. 31: Steeves, R., and F. Lilly Interactions between host and viral genomes in mouse leukemia. Annu. Rev. Genet. 11: Tennant, R. W., J. A. Otten, A. Brown, W. K. Yang, and S. J. Kennel Characterization of Fv-1 host range strains of murine retroviruses by titration and p3 protein characteristics. Virology 99: Yoshikura, H Ultraviolet inactivation of murine leukemia virus and its assay in permissive and non-permissive cells. Int. J. Cancer 11: Downloaded from on November 15, 218 by guest