Differential Effects of C-Terminal Molecular Tagged Integrase on Replication Competent Moloney Murine Leukemia Virus

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1 Virology 274, (2000) doi: /viro , available online at on Differential Effects of C-Terminal Molecular Tagged Integrase on Replication Competent Moloney Murine Leukemia Virus Jennifer A. Seamon, Monica Adams, Sonali Sengupta, and Monica J. Roth 1 Department of Biochemistry, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey Received March 31, 2000; returned to author for revision April 11, 2000; accepted May 27, 2000 Moloney murine leukemia virus (M-MuLV) proviruses carrying integrase (IN) protein tagged either with a simian virus 40 (SV40) nuclear localization signal (NLS) or various antigenic epitopes were generated. Hexahistidine (His 6 ), hemagluttinin (HA), or two consecutive HA sequences (2XHA) were fused to the C-terminus of IN as antigenic markers. These epitopetagged IN proteins were stably expressed through multiple rounds of infection. The IN-His 6, IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His 6 and HA, respectively. An M-MuLV provirus encoding the SV40 large T antigen NLS fused to IN at the same position as the epitope tags was also passaged through cells. In contrast to the stability of the epitope tags, the SV40 NLS sequence was rapidly mutated by a frameshift mutation that introduced negatively charged amino acids into the basic NLS. The instability of the NLS suggests that the strong nuclear localization of the IN-SV40 NLS may have detrimental effects on virus assembly. These observations have implications for studying nuclear transport properties of M-MuLV and for engineering a murine-based retroviral vector for gene therapy Academic Press INTRODUCTION 1 To whom reprint requests should be addressed. Fax: (732) roth@waksman.rutgers.edu. Productive retroviral infections require the stable integration of the reverse-transcribed viral genome into the host chromosome. This process is mediated by the enzymatic activity of the virally encoded integrase (IN) protein. Upon entry into the cytoplasm of the host cell, reverse transcription of the viral RNA genome into double-stranded DNA occurs within a large nucleoprotein complex derived from the core of the infecting virion, termed preintegration complexes (PICs). The resulting double-stranded viral DNA is maintained in this stable nucleoprotein complex, which localizes to the nucleus. The protein composition and the architecture of the subviral PICs are being extensively studied. Murine leukemia virus (MuLV) PICs retain capsid (CA) protein derived from the virion core (Bowerman et al., 1989). In addition, the intracellular complexes shown to mediate Moloney MuLV (M-MuLV) reverse transcription are composed of the CA, IN, and reverse transcriptase (RT) proteins (Fassati and Goff, 1999). The organization of the human immunodeficiency virus type 1 (HIV-1) PICs, however, appears to differ. IN, matrix (MA), and RT proteins were found present in HIV-1 PICs, with no CA associated (Farnet and Hazeltine, 1991; Miller et al., 1997). Host factors for both MuLV and HIV-1 have also been shown to associate with PICs and to play a role in the integration process (Chen and Engelman, 1998; Farnet and Bushman, 1997; Kalpana et al., 1994; Lee and Craigie, 1998; Li et al., 1998). The subcellular trafficking of PICs varies among the different retroviruses. HIV-1 is able to infect nondividing cells (Lewis et al., 1992; Weinberg et al., 1991). Three HIV-1 proteins have been implicated in the active transport of HIV-1 PICs into the nucleus: IN, MA, and Vpr (Bukrinsky et al., 1993; Gallay et al., 1997; Heinzinger et al., 1994). The IN protein contains a nuclear localization signal (NLS) that interacts with members of the karyopherin family of nuclear import receptors (Gallay et al., 1997). Two potential NLS sequences within the HIV-1 MA have been identified (Bukrinsky et al., 1993; Nadler et al., 1997). There are, however, conflicting reports regarding the ability of these basic regions to function in post-entry events (Bukrinsky and Haffar, 1999; Bukrinsky et al., 1993; Dupont et al., 1999; Fouchier et al., 1997; Freed et al., 1995; Gallay et al., 1996; Reil et al., 1998). The Avian sarcoma virus (ASV) IN similarly contains a nuclear targeting signal, which is proline rich and basic in nature (Kukolj et al., 1997). In contrast, studies with M-MuLV do not reveal an active nuclear transport process. Nuclear accumulation of M-MuLV viral DNA increases only after infected cells pass through mitosis (Roe et al., 1993). These differences in transport may reflect the differences in the composition of the PICs or the lack of appropriate targeting signals for M-MuLV. In this study, we report the construction of replicationcompetent M-MuLV clones carrying IN fusions to various /00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved. 412

2 MOLECULAR TAGGING OF M-MuLV INTEGRASE PROTEIN 413 FIG. 1. Schematic diagram illustrating insertion of peptides into the C-terminus of M-MuLV IN protein within the context of replication competent proviruses. (A) Insertions within IN were performed in the vector pnca-c-xn-su8, encoding the complete M-MuLV proviral DNA genome, including the long terminal repeats (LTR) and the gag, pol, and env genes. The plasmid contains an SuIII tyrosine suppressor trna, which was inserted into a nonessential region of the 3 LTR (in31suiii; Lobel et al., 1985). The IN protein is encoded at the 3 -terminus of the pol gene of M-MuLV. The env gene products overlap the IN protein and are expressed from an alternate reading frame. The vertical line indicates position 6215 (Shinnick et al., 1981), where the XbaI site was destroyed by insertion of an NotI linker (Roth, 1991). Each of the peptide fusions was inserted individually at this position, immediately upstream of a TGA stop codon. (B) The expanded sequence of the tagged C-terminus of IN is shown. The DNA sequence of each of the molecular tags is underlined with the amino acid sequence under each codon. The in-frame TGA stop codon is marked in bold. molecular tags. Furthermore, we show that these M- MuLV IN C-terminal fusions can be incorporated into viral particles. Similar studies have shown that HIV-1 IN can be epitope tagged within the context of a replicationcompetent virus (Petit et al., 1999). Extending this, the work presented in this study examines the effects of fusing a simian virus 40 (SV40) large T antigen NLS to M-MuLV IN. Interestingly, the virus rapidly reverted and disrupted the basic nature of the NLS sequence. This indicates an effective selection against insertion of a basic NLS within IN. This study generates a model system that can examine the influence of alternative NLS sequences on the nuclear transport properties of M- MuLV. The results provide insights into the mechanisms developed by retroviruses to maintain the balance between the export and import of viral particles and have broad implications regarding the complexities evolved to allow cell-cycle independent viral entry. RESULTS Generation of IN-tagged viral clones To gain a better understanding of the viability and tolerance of modified IN proteins within a replication competent virus, four ecotropic M-MuLV proviral clones were constructed with molecular tags fused to IN (Fig. 1). The vector used, pnca-c-xn-su8, encodes a novel NotI restriction site within IN (Roth, 1991), plus a suppressor trna in the 3 LTR (Lobel et al., 1985). As previously described, the NotI linker at the XbaI site (position 6215; Shinnick et al., 1981) of IN positions a TGA stop codon immediately downstream of the linker. This yields a viable virus with a 23-amino-acid truncation of the IN protein (Roth, 1991). Because the first 19 amino acids of the M-MuLV env gene product overlap the C-terminus of IN in an alternate reading frame, this approach allows for truncation of the IN protein without generating large deletions within the 5 env leader sequence. In the present study, the unique NotI site of pnca-c- XN-SU8 served as the insertion site for oligonucleotides encoding either a hexahistidine (His 6 ), a hemagluttinin epitope (HA), two consecutive hemagluttinin epitopes (2XHA), or an SV40 large T antigen NLS (Fig. 1A). This allowed for the expression of a truncated IN protein C-terminally fused to a particular molecular tag. The sequence of each of the molecular tags is shown in Fig. 1B. For each of the insertions, the TGA stop codon immediately 3 of the NotI site is maintained in-frame with the IN coding sequence and should result in the deletion of the IN C-terminus (Fig. 1B). Effects on the viral life cycle of the molecular tagged clones The effects of the various molecular tags on the M- MuLV viral life cycle were examined. Plasmids contain-

3 414 SEAMON ET AL. FIG. 2. Time course of infection of the tagged IN viruses. (A and B) Plasmid DNA containing the complete proviral M-MuLV bearing the specific tagged IN sequences was transiently introduced into D17 cells, stably transfected with the ecotropic MCAT receptor (pjet). At various days posttransfection, supernatant from cultured medium was collected and assayed for RT activity to monitor viral production. The specific tagged IN viruses are indicated above the lanes. The number of days after the DNA was introduced into the cells is indicated at the left. (C) Virus collected from the various tagged viruses, wild-type and mock cell lines was normalized with respect to RT activity and applied to a fresh culture of pjet cells. RT assays, as described for A and B, were performed on culture supernatants at the indicated days after infection. The arrows indicate the point where the cells were passed. The IN tags are indicated above the lanes. The number of days postinfection is indicated at the left. ing proviral DNA encoding the tagged IN proteins were separately introduced into D17 cells expressing the MCAT ecotropic receptor (pjet). Transient expression of the plasmid DNA allows for assembly and release of viral particles and subsequent reinfection. Viable virus can then spread throughout the plate. Viral propagation was monitored by the release of RT activity into the media. In the wild-type M-MuLV (pnca-c) transfected culture, RT activity was detected by approximately day 7 (Fig. 2A). The M-MuLV IN tagged viral clones IN-His 6, IN-HA, and IN-SV40 NLS showed a minor delay in viral spreading compared with the wild type, with detection of RT activity by approximately day 9 (Fig. 2A). Levels equivalent to the parental MuLV can be reached after passage for approximately 2 weeks (data not shown). This result indicates that the molecular tagging of the truncated IN does not grossly impede function in vivo. In contrast, the IN-2XHA molecular tag produced a much longer delay in the appearance of virus in the culture supernatants. RT activity was not detected until day 25 (Fig. 2B). Virus from producer cells, established by the transient transfection, were examined for signs of reversion. Equal amounts of wild-type and tagged viruses, normalized with respect to RT activity, were used to infect fresh pjet cells. Infection by the IN-2XHA progeny viruses remained delayed relative to wild type, with RT activity detected 11 days after the wild-type virus (Fig. 2C). This indicates that a revertant of the IN-2XHA virus with improved kinetics of infection was not selected during viral passage. Interestingly, the rates of release of progeny IN-His 6, IN-HA, and IN-SV40 NLS viruses were similar to that of wild type (Fig. 2C). The minor delayed growth of these viruses was not maintained upon infection of fresh cells. Analysis of viral proteins To ensure that the tagged IN proteins were stably expressed and present in virions, viral proteins from the viable tagged viruses were examined. Producer cells were metabolically labeled, and viral particles were isolated and immunoprecipitated with serum that specifically recognizes RT/IN proteins (Fig. 3A). In all cases, immunoprecipitation with this serum resulted in identification of both RT and IN. Wild-type RT (p80) and IN (p45) are shown in Fig. 3A (lanes 4 and 8). The immunoprecipitated IN-His 6, IN-HA, and IN-2XHA proteins migrated at their predicted molecular mass. The IN-His 6 and IN-HA proteins were predicted to migrate faster than the wild-type IN; each truncate the C-terminal 23 amino acids of IN and append 13 and 15 nonvirally encoded amino acids, respectively (Fig. 3A, lanes 2 and 6, compared with wild type, lanes 4 and 8, respectively). IN- 2XHA migrated at a similar position to wild-type IN (Fig. 3A lanes 7 and 8), the net result of the C-terminal 23 amino acid truncation, plus the addition of 23 nonvirally encoded amino acids by the linker-insertion. Insertion of these molecular tags therefore resulted in the production of stable IN proteins. The ability of the modified IN proteins to be recognized by the antibodies specific to the inserted epitopes was examined. IN-His 6, IN-HA, and IN-2XHA proteins, purified from labeled virus, were immunoprecipitated with antibodies against His 6 and HA, respectively. Immunoprecipitation with the antibodies against His 6 resulted in the identification of the 37-kDa protein recognized by the RT/IN serum (Fig. 3B, lanes 9 11). Similarly, antibodies against HA that recognize the HA epitope YPYDVPDYA

4 MOLECULAR TAGGING OF M-MuLV INTEGRASE PROTEIN 415 FIG. 3. Analysis of viral proteins. (A and B) Proteins were metabolically labeled, and viral particles were isolated. Labeled viral proteins were normalized with respect to RT activity. Proteins were immunoprecipitated with either anti-pol serum (Tanese et al., 1986), anti-his 6 monoclonal antibodies, or anti-ha polyclonal antibodies. The specific viral stocks are indicated at the top along with the antibodies used for immunoprecipitation. The positions of RT, wild-type IN, and tagged IN proteins are indicated at the right. The positions of migration of the prestained protein standards and their molecular masses are indicated to the side of each panel. specifically immunoprecipitated the IN-HA- and IN- 2XHA-labeled proteins (Fig. 3B, lanes 12 15). Interestingly, the antibodies to both the His 6 and HA epitopes coimmunoprecipitated the tagged IN and RT, indicating an RT IN interaction within the virus. This interaction was previously observed with antibodies to IN (Hu et al., 1986). Although the IN-His 6, IN-HA, and IN-2XHA proteins migrated at their predicted molecular weights, immunoprecipitation of the labeled IN-SV40 NLS virus surprisingly yielded protein that ran at a slower mobility than predicted (Fig. 3A). The IN-SV40 NLS protein was predicted to run at a faster mobility than the wild-type IN because it is the net result of the 23-amino-acid C- terminal truncation of IN, plus only 11 amino acids encoding the SV40 NLS insertion (Fig. 3A, lanes 3 and 4). The detection of this shift suggested that the virus had reverted. Sequence analysis of unintegrated viral DNA To investigate the cause of the aberrant mobility of the IN-SV40 NLS protein on an SDS PAGE gel, unintegrated, FIG. 4. Sequence analysis of unintegrated viral DNA from cells freshly infected with IN-SV40 NLS. Unintegrated viral DNA was isolated from freshly infected cells and sequenced. The sequence of the reverted NLS is shown in bold with the out-of-frame TGA stop codon boxed. extrachromosomal viral DNA was isolated from freshly infected cells and sequenced. Sequence analysis of the unintegrated viral DNA from the IN-SV40 NLS virus revealed an insertion of an adenine (A) within a stretch of six A residues encoding the SV40 NLS sequence. The shift in the reading frame introduced negatively charged amino acids within the basic NLS and restored the original reading frame of the C-terminus of IN (Fig. 4). No other base changes within the IN sequence were observed upon complete sequencing of the tagged IN (data not shown). Therefore, the restoration of the full-length IN protein and the addition of the 11 nonvirally encoded amino acids account for the slow mobility of the IN-SV40 NLS protein on an SDS PAGE gel (Fig. 3A, lane 3). Although the parental IN-SV40 NLS virus must have been viable to allow for the rapid generation of the altered virus, the viruses containing the disrupted NLS sequence were selected and subsequently propagated. The original IN-SV40 NLS proviral construct was transfected in triplicate. Identical reversion events were observed in all three cases. Sequence analysis of the isolated viral DNA from IN- His 6, IN-HA, and IN-2XHA viruses showed that the tags were maintained. Furthermore, complete sequence analysis of the entire IN gene of the IN-2XHA virus revealed no changes (data not shown). Therefore, these epitopes were stably maintained within IN, despite the differences in their kinetics of viral infection. DISCUSSION The basic residues of the well-characterized NLS of the SV40 large T antigen have been shown to direct

5 416 SEAMON ET AL. nuclear entry of cytosolic proteins when incorporated into fusion proteins (Kalderon et al., 1984b). Furthermore, these residues are recognized by the cytosolic nuclear receptors known as importins, which dock the NLScontaining proteins at the nuclear pore complex (Gorlich, 1997). In this study, we described that the expression of the replication competent provirus with truncated IN C- terminally fused to the biologically relevant SV40 NLS led to a reversion event, which disrupted the basic NLS sequence and restored the full expression of the C- terminus of IN. Our data suggest that the reversion event took place due to the nature of the NLS sequence and not the insertion site used to generate the modified IN. Fusion of His 6, HA, or 2XHA epitopes to the truncated C-terminus of IN did not result in the disruption of the molecular tags. Furthermore, the parental IN-SV40 NLS virus must have been viable to allow for the rapid generation of the altered virus. The virally encoded RT is a major contributor to variation in retroviral genomes due to the lack of exonucleolytic proofreading activity of the enzyme. Results from our studies of the transient expression of the SV40 NLS tagged virus revealed an insertion of an adenine (A) within a stretch of six A residues of the NLS. It is interesting that the reversion consistently occurred through an insertion, rather than deletion of the NLS sequence. The experiment was performed in triplicate; each independent isolate resulted in an identical insertion. Complete sequencing of an entire IN gene did not detect any other change in either the IN or the NLS. Preliminary data indicate that a second NLS sequence, based on the nucleoplasmin NLS, reverted using a similar mechanism within an A 6 sequence (J.S., unpublished observations). One likely mechanism that could account for disruption of the NLS at this particular site is slippage of the polymerase domain of RT within the stretch of six A residues, yielding a frameshift. In a single replication cycle for spleen necrosis virus (SNV), a majority of the frameshift mutations observed occurred in runs of identical nucleotides (Kim et al., 1996), with an increase in mutation frequency occurring with each increase in length of the nucleotide run (Pathak and Temin, 1990). Slippage of RNA polymerase is not uncommon in viral systems. It is suggested that for late transcripts of vaccinia virus, the poly(a) leaders on their 5 ends result from slippage of the RNA polymerase. The slippage occurs within the highly conserved TAAAT element within the late promoters and requires three consecutive A residues (Davison and Moss, 1989). The rapidity with which the reversion event took place makes it difficult to examine which step in the viral life cycle was inhibited by the insertion of the SV40 NLS sequence. There are several potential driving forces that could account for the reversion. One possibility is that the basic NLS sequence may function as a nucleic acidbinding site. IN protein is required to interact specifically with the termini of viral LTRs and nonspecifically with the target DNA. Insertion of an alternative DNA binding site might interfere with the precise coordination of protein DNA required for retroviral integration. Alternatively, the restoration of the full expression of the C-terminus of IN could also be a driving force in the reversion event. Sequence alignment with various MuLVs showed that natural selection appears to act on conserving the C-terminal domain of IN, rather than on the overlapping env gene. This suggested a functional role for this region of IN (Ey et al., 1997). The data indicate, however, that truncation of the IN C-terminus does not impede the virus. Viral infection was not grossly inhibited for the His 6 and HA tagged proviral constructs, which lack the C-terminus of IN. Furthermore, the parental construct, pin6215a, which identified the C-terminus of IN as nonessential, and pnca-c-xn-su8 showed no delay in viral spreading compared with wild type (Roth, 1991; J.S., unpublished observations). The disruption of the NLS sequence by these viruses most likely reflects the regulation of viral export versus viral entry. Retroviral IN is expressed and incorporated into virions as part of the Gag-Pol polyprotein precursor. During retroviral assembly, precursor Gag-Pol proteins are transported to the cytoplasmic face of the plasma membrane, where they mediate budding. It is possible that the strong NLS interfered with the targeting of the Gag-Pol precursor to the cell membrane by directing the precursor back to the nucleus. Characterization of the SV40 NLS indicates that substitution of amino acids within the basic tract of the NLS results in the cytoplasmic localization of large-t antigen and synthetic peptides (Chelsky et al., 1989; Kalderon et al., 1984a). The reversion identified in this study similarly disrupted the basic tract of the SV40 NLS. Furthermore, preliminary data indicate a second IN-NLS, based on the nucleoplasmin NLS, also reverted to disrupt the basic nature of the sequence (J.S., unpublished observation). It does not appear that appending an SV40 NLS onto IN merely affected the stability of the protein. IN-SV40 NLS can be stably expressed on modification of the codon use of the SV40 NLS (J.S., unpublished observations). It is interesting that the putative NLS within MA of HIV-1 is overlapping with a nuclear export sequence (Dupont et al., 1999). Furthermore, it is important to note that the NLS sequences identified within the HIV-1 and ASV IN proteins (Gallay et al., 1997; Kukolj et al., 1998) have not been simple basic NLS sequences, such as found in the SV40 T antigen. We predict that the selective forces on these alternative NLS sequences may not be present if these sequences were substituted within M-MuLV IN. This assay system is being developed to analyze NLS strength with the goal of altering the cell-cycle dependency of M-MuLV. It is conceivable that the delay in viral spreading introduced by the insertion of the 2XHA tag could have

6 MOLECULAR TAGGING OF M-MuLV INTEGRASE PROTEIN 417 resulted from alterations in IN structure because the 2XHA tag was the largest sequence C-terminally fused to the truncated IN. The M-MuLV proviral clones containing the smaller His 6 and HA molecular tags showed no significant delay in viral spreading. We have not ruled out the possibility that the observed delay is due to insufficient DNA synthesis or integration in vivo. In summary, the results of this study indicate that C-terminal IN fusions, within a replication competent M-MuLV provirus, can be stably expressed through multiple rounds of replication. In addition, these fusion proteins can be incorporated into viral particles. This is in contrast to observations made in an ASV fusion protein strategy, where fusion of LexA DNA binding domains onto the C-terminus of ASV IN, in the context of an infectious virus, were shown to be genetically unstable (Katz et al., 1996). Tags placed within HIV-1 IN were useful in demonstrating the presence of IN oligomers within virions (Petit et al., 1999). We report the fusion of the active transport sequence, the SV40 NLS, into M- MuLV IN. The results of such a fusion suggest the possibility that infectious M-MuLV encoding IN C-terminally fused with alternative NLS sequences, such as those found in HIV-1 or ASV IN proteins (Gallay et al., 1997; Kukolj et al., 1998), may generate tagged M-MuLV with altered nuclear transport properties. The generation of M-MuLV with altered transport properties would have great implications in understanding the relationship between M-MuLV infection and the cell cycle, and in engineering murine-based retroviral vectors. Plasmid construction MATERIALS AND METHODS Construction and analysis of the viable replication competent pin6215, in which a NotI linker was inserted within the XbaI site (position 6215 (Shinnick et al., 1981)) of IN, were previously described (Roth, 1991). A suppressor trna was inserted into a nonessential region of the 3 LTR (Lobel et al., 1985). The resulting plasmid, pnca- C-XN-SU8, was digested with NotI, and the oligonucleotides encoding each of the molecular tags were ligated at a 1000-fold molar excess to the plasmid. Oligonucleotides, encoding for the sense and antisense sequences of each of the molecular tags, were purchased from the University of Medicine and Dentistry of New Jersey Biochemistry Department Synthesis Facility. The annealed oligonucleotides contain NotI 5 extensions. The oligonucleotides that encode the hexahistidine (His 6 ) tag are as follows: 8066-sense, 5 -GGCCGGCCATCACCATCACCAT- CACTT-3 ; and 8065-antisense, 3 -CCGGTAGTGGTAGTGG- TAGTGAACCGG-5. Oligonucleotides used to generate the HA tag are 8067-sense, 5 -GGCCGGCTACCCATACGATGT- TCCAGATTACTT-3 ; and 8068-antisense, 3 -CCGATGGG- TATGCTACAAGGTCTAATGAACCGG-5 ; they are based on the HA1 strain of influenza. Oligonucleotides 8405-sense 5 -GGCCCCAAAAAAGAAGAGAAA-3 and 8406anti-sense 3- GGTTTTTTCTTCTCTTTCCGG-5 were used to encode for the SV40 large T antigen NLS. The sequence of each of the tagged regions up to and including the stop codon was confirmed by performing dideoxynucleotide sequencing, using AmpliTaq DNA polymerase (Perkin Elmer) and 5 labeled primer (6992) 5 -AGTACTGACCCCTCTG-3 (nucleotides , M-MuLV; Shinnick et al., 1981). The sequences of the 2XHA oligonucleotides are sense, 5 -GCGTACCCATACGATGTTCCAGATTACTACCCAT- ACGATGTTCCAGATTAC-3 ; and 8950-antisense, 3 -CGCA- TGGGTATGCTACAAGGTCTAATGATGGGTATGCTACAAGG- TCTAATG-5. The blunt NaeI site generated by the annealed HA oligonucleotides was used to insert the 2XHA oligonucleotides into pnca-c-xn-su8 containing the HA tag. Although this should have yielded three in-frame HA sequences, only two in-frame tags were generated. The wild-type M-MuLV proviral plasmid pnca-c was previously described (Felkner and Roth, 1992). Cell culture The MCAT ecotropic receptor (pjet; Albritton et al., 1989) was stably transfected into D17 cells (O Reilly and Roth, 2000) grown in Dulbecco s modified Eagle s medium (GIBCO BRL, Grand Island, NY) supplemented with 7.5% fetal calf serum, glutamine (0.29 mg/ml), penicillin (5 U/ml), streptomycin (5 g/ml), and G418 (400 g/ml) to maintain the ecotropic receptor. Plasmid DNA (0.5 g) containing the complete proviral M-MuLV bearing the specific tagged IN sequences was transiently introduced into 10 5 cells/6-cm dish in the presence of DEAE-dextran (McCutchan and Pagano, 1968). RT assay At various days post-transfection, supernatant from cultured medium was collected. 10- l aliquots of the media were used in an RT assay as previously described (Goff et al., 1981). Metabolic labeling and isolation of viral proteins Proteins were metabolically labeled with 150 Ci of [ 35 S]methionine TRANS[ 35 S]LABEL (ICN) and labeled viral particles were isolated as previously described (Felkner and Roth, 1992). Viral proteins were immunoprecipitated, as described (Gray and Roth, 1993) with either 8 l of anti-pol serum (Tanese et al., 1986), 18.3 g (1:62 dilution) of anti-his 6 monoclonal antibodies (catalog H-1029; Sigma Chemical Co., St. Louis, MO), or 1:150 dilution of anti-ha polyclonal antibodies (catalog PRB- 101C; Babco). Isolation and characterization of M-MuLV viral clones Unintegrated viral DNA was isolated using the method of Hirt (Hirt, 1967). DNA from the HIRT extract was PCR

7 418 SEAMON ET AL. amplified as previously described (Smith et al., 1997) using primers (3807) 5 -GCCGTTAAACAGGGA-3 (nucleotides , M-MuLV; Shinnick et al., 1981) and (6877) 5 -AGTTGTCTGGTCTAGC-3 (nucleotides , M-MuLV; Shinnick et al., 1981). The PCR products were gel isolated, purified by glass powder (BioLabs), drop dialyzed on m filter paper in water, and sequenced by the dideoxynucleotide method. ACKNOWLEDGMENTS This research is supported by National Institutes of Health (NIH) Grant R21-DK54374 and R01CA76545 to M.J.R. J.S. is supported in part by NIH Predoctoral Training Grants T32-GM8360 and T32-AI7403. We thank Christina Miller for her assistance. REFERENCES Albritton, L. M., Tweng, L., Scadden, D., and Cunningham, J. M. (1989). A putative murine retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57, Bowerman, B., Brown, P. O., Bishop, J. M., and Varmus, H. E. (1989). 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