Capsid Processing Requirements for Abrogation of F
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病菌学杂志 2005年第16期
Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
ABSTRACT
Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.
INTRODUCTION
It is becoming increasingly clear that mammalian cells can express a variety of factors that limit retroviral replication. One class of these factors targets the retroviral capsid (CA) protein and blocks replication at a stage postentry but prior to integration of the viral genetic material into the cell genome. Members of this family are known as restriction factors (1, 12). The best-characterized member of this family is the mouse Fv1 (19) gene, which determines susceptibility to infection by murine leukemia virus (MLV). Fv1 has two major alleles, known as Fv1n and Fv1b, whose restriction characteristics determine the host range of different MLV strains, allowing the division of MLV into a number of classes, two of which are known as N tropic and B tropic (16). An Fv1n/n mouse (or cell line) restricts the B-tropic virus but is permissive to N-tropic MLV. An Fv1b/b mouse restricts the N-tropic virus but not B-tropic MLV. Resistance is semidominant; heterozygous animals restrict both classes of virus. NB-tropic viruses are relatively insensitive to Fv1 restriction (14). A similar form of MLV restriction exists in human cell lines. Restriction is comparable to that of the B allele of Fv1 in that the N-tropic virus is restricted while the B-tropic virus is not. This restriction factor has been named Ref1 (34). Two members of the TRIM family of proteins, Trim5 and Trim1, have been implicated as components of this restriction (15, 17, 39). Trim5 from several primate species can also restrict HIV-1 (22, 29, 32).
The major determinant of MLV restriction has been mapped to the CA protein. A single amino acid change within the capsid (R110E) will convert an N-tropic virus to a B-tropic virus and a Ref1-sensitive MLV to a Ref1-resistant virus (18, 34). Despite this, it has so far been impossible to show a direct interaction between CA and Fv1 using standard methods, such as yeast two-hybrid or coimmunoprecipitation techniques (unpublished data). However, restriction by Ref1 or Fv1 can be overcome when infecting cells at a high multiplicity of infection (7, 35). Further, pretreatment of cells with restricted virus allows unrestricted infection by a second, challenge virus (3). This suggests that restriction factor is limiting and can be saturated, or "abrogated," by restricted virus. Consistent with this proposition, natural levels of Fv1 expression appear very low (40). Abrogation therefore provides the basis of a method for studying the interaction between CA and restriction factors.
Despite the fact that the restriction factor can be abrogated by exposure to restricted virus at a high multiplicity of infection, chronically infected cell lines in which high levels of restricted Gag polyprotein are expressed can still restrict incoming virus (8, 16). Two explanations, not necessarily mutually exclusive, might be advanced to explain this apparent paradox. First, Gag processing and/or assembly might be required for CA recognition by Fv1. Only after a virus is released from the producer cell is the viral Gag cleaved into its four constituent parts—matrix (MA), p12, CA, and nucleocapsid (NC)—by the viral protease (PR), thereby leading to the condensation of the CA component to form a spherical core containing the RNA genome. It does not seem unreasonable to suppose that Fv1 interacts only with the mature, multimerized form of CA. Alternatively, it seems possible that the intracellular localization of CA during viral egress and ingress plays a critical role. Active Fv1 is found in association with tubules of the trans-Golgi network (40), and it may be that Gag polyprotein never meets Fv1 during its outgoing transit through the cell. In this report we describe experiments designed to address these possibilities, focusing on establishing the relationship between Gag processing and abrogation.
MATERIALS AND METHODS
Cell culture and virus preparations. 293T, Mus dunni, BALB/3T3, NIH 3T3, and TE671 cell lines were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and penicillin/streptomycin (Sigma). Viruses were prepared by transient transfection of 293T cells using the Promega Profection mammalian transfection system, typically with 7 μg gag-pol expression vector, pCIG3 N, B, pHIT 60 (30), or derivatives described below; 7 μg of vesicular stomatitis virus G protein expression vector, pczVSV-G (25), and 7 μg of either pLNCG (encoding enhanced green fluorescent protein [eGFP]) (39) or pHIT111 (lacZ) (30) or pLIB-NCA/BCA-puro (see below).
Generation of mutant gag-pol expression vectors. Mutants of pCIG3 N and B plasmids were made by PCR site-directed mutagenesis using PfuTurbo DNA polymerase (Stratagene) according to the manufacturer's protocol with the following oligonucleotides: protease D32L (PR–), forward, 5'-CCGTCACCTTCCTGGTGCTTACTGGGGCCCAACACTCC-3', and reverse, 5'-GGAGTGTTGGGCCCCAGTAAGCACCAGGAAGGTGACGGG-3'; reverse transcriptase D224E (RT–), forward, 5'-CCTGCTACAGTACGTGGAGGACATACTACTGGCC-3', and reverse, 5'-GGCCAGTAGTATGTCCTCCACGTACTGTAGCAGG-3'; MA-p12 (MAxp12), forward, 5'-CCCCGATCTGCCCTTGATCCTGCTCTTACCCC-3', and reverse, 5'-GGGGTAAGAGCAGGATCAAGGGCAGATCGGGG-3'; p12-CA (p12xCA), forward, 5'-CCACCTCTCGGGCTGACCCACTCCGTTTGGGG-3', and reverse, 5'-CCCCAAACGGAGTGGGTCAGCCCGAGAGGTGG-3'; CA-NC (CAxNC), forward, 5'-GGAATGAGCAAACTTAGGGCCACCGTAGTTAG-3', and reverse, 5'-CTAACTACGGTGGCCCTAAGTTTGCTCATTTC-3'; CA D54A (D54A), forward, 5'-CAGCCCACCTGGGATGCCTGCCAGCAATTATTAG-3', and reverse, 5'-CTAATAATTGCTGGCAGGCATCCCAGGTGGGCTG-3'.
The sequences of the mutations introduced were verified by DNA sequencing.
Abrogation experiments. In all cases, cells were plated at a density of 4 x 104 per well in a 12-well plate 24 h prior to transduction. These were pretreated with 500 μl of the appropriate virus-like particles (VLP), at concentrations given below, for 3 h, before challenge with a fixed amount of either N- or B-tropic virus carrying the pLNCG (eGFP) vector. The percentage of transduced cells was analyzed by fluorescence-activated cell sorter (FACS) analysis after 3 days.
Derivation of single-cell clones expressing CA. To insert a foot-and-mouth disease virus (FMDV) 2A protease between p12 and CA, thereby allowing intracellular expression of properly processed CA, the gag region of pCIG3 N and B was amplified by PCR in two sections. Primers were designed to produce an EcoRI-MA-p12-(N-terminal 2A)-HindIII PCR product and a HindIII-(C-terminal 2A)-CA-MfeI PCR product. The following primer sets were used: for MA-p12-2A, forward, 5'-GCGAATTCACATGGGACAGACCGTAACC-3', and reverse, 5'-GCAAGCTTAAGAAGGTCAAAATTCAACAGAAAGCCCCGAGAGGTGGTGG-3'; 2A-CA, forward, 5'-GCAAGCTTGCGGGAGACGTCGAGTCCAACCCCGGGCCACTCCGTTTG-3', and reverse, 5'-CGCAATTGTTACAAAAGTTTGCTCATTTCTCTA-3'. Each product was cloned into pCRII-TOPO (Invitrogen). These plasmids were then digested with either EcoRI/HindIII for the MA-p12-2A insert or HindIII/MfeI for the 2A-CA insert. A triple ligation was performed with these two inserts and the pIRESpuro vector (Clontech) (previously cut with EcoRI), creating MA-p12-2A-CA-IRES-puro. The full construct was excised with EcoRI and StuI and ligated into pLIB (Clontech), previously cut with the same enzymes, producing pLIB-NCA-puro and pLIB-BCA-puro. Viral vectors carrying pLIB-NCA-puro and pLIB-BCA-puro were prepared following transfection of 293T cells. Serial dilutions of each virus were prepared (10–1, 10–2, 10–3, and 10–4), with 100 μl of each used to infect a 6-cm plate with 5 x 105 NIH 3T3, BALB/3T3, M. dunni, or TE671 cells. After 2 days, puromycin was added to the media at a concentration of 2 μg/ml.
Two weeks later, plates with separate colonies could be identified for each cell line and expression construct. Three colonies of each type were isolated with cloning rings (Sigma) and grown as individual cultures. Cells were screened for CA expression by Western blot analysis.
Western blotting. Virions were pelleted by centrifugation at 55,000 rpm for 90 min in a Beckman TLA 120.1 rotor, washed once with phosphate-buffered saline (PBS), suspended in sodium dodecyl sulfate (SDS)/loading dye containing 10 mM dithiothreitol (DTT), and heated to 90°C for 5 min before electrophoresis on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. Proteins were transferred to polyvinylidene difluoride membranes (Millipore) and probed with monoclonal anti-CA (4) or polyclonal anti-p12 antibodies. Cell lysates from N-tropic CA/B-tropic CA (NCA/BCA)-expressing single-cell clones were prepared by adding 500 μl triple detergent lysis buffer (28) to confluent cells in a 6-cm dish and cleared by centrifugation. Following the addition of SDS/loading dye containing 10 mM DTT to the supernatant, samples were heated to 90°C for 5 min, and 35 μl was loaded onto a 12% SDS-PAGE gel and processed as described above.
CA enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G fractions from goat anti-CA polyclonal antisera (Viromed) were purified on a protein A column (13). This material was biotinylated using the Immunoprobe Biotinylation kit (Sigma) according to the manufacturer's guidelines. Wells from a 96-well Reacti-Bind streptavidin plate (Pierce) were washed three times with 250 μl PBS, 0.1% bovine serum albumin, 0.05% Tween before being incubated overnight at 4°C with 100 μl of 25 μg/ml biotinylated anti-CA (diluted in wash buffer). Plates were washed three more times before the addition of 100 μl of viral sample, pretreated with 1% NP-40 for 15 min at 37°C to disrupt viral membranes, and then incubated at room temperature for 1 h. After three further washes, rat monoclonal anti-CA antibody (100 μl) was then added at a concentration of 10 μg/ml (diluted in wash buffer), and the plates were incubated for a further 90 min at room temperature. After three more washes, 100 μl of monoclonal goat anti-rat immunoglobulin G conjugated to calf intestinal phosphatase (Pierce) was added at a concentration of 6 μg/ml and incubated at room temperature for 1 h. This was then washed 10 times with wash buffer. Binding of this antibody was detected using the Phosphatase Substrate kit (Pierce) according to the manufacturer's protocol. A405 was measured using a Thermo Labsystems Multiskan Ascent spectrophotometer. Concentrations were determined from the mean A405 of three replicates. Standard curves were obtained with N-terminally His-tagged CA purified from Escherichia coli (21).
Immunoprecipitation and fingerprinting by matrix-assisted laser desorption ionization mass spectrometry. M. dunni cells expressing CA from N-tropic MLV were lysed using a nondenaturing lysis buffer, and the CA was immunoprecipitated using rat monoclonal anti-pCA antibody bound to protein G beads (5). Beads were then resuspended in SDS/loading dye containing 10 mM DTT and then incubated at 90°C for 5 min to detach the bound protein. Following centrifugation, the supernatant was loaded onto a 12% SDS-PAGE gel. After electrophoresis, gels were stained with Simply Blue Safe Stain colloidal Coomassie (Invitrogen), and the band at 30 kDa was excised, reduced with dithiothreitol, and alkylated using iodoacetamide. Gel slices were dried and reswollen in a sufficient volume to cover 4 ng/μl endoproteinase Asp-N (sequencing grade; Roche) in 5 mM ammonium bicarbonate. After overnight digestion at 32°C, the supernatant was acidified by the addition of a 1/10 volume of 4% trifluoroacetic acid. Peptide mass fingerprinting was performed using a Reflex III matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), equipped with a nitrogen laser and a Scout-384 probe, to obtain positive ion mass spectra of digested protein with pulsed ion extraction in reflectron mode. An accelerating voltage of 26 kV was used with detector bias gating set to 2 kV and a mass cutoff of m/z 650. Matrix surfaces were prepared using recrystallized -cyano-4-hydroxycinammic acid and nitrocellulose, using the fast evaporation method (37) Digestion supernatant (0.4 μl) was deposited on the matrix surface and allowed to dry prior to desalting with water. Peptide mass fingerprints were compared to the nonredundant protein database placed in the public domain by the National Center for Biotechnology Information using the program MASCOT (24).
Immunofluorescence. Cells were plated at a density of 2 x 104 per well on a glass coverslip in a 12-well plate 24 h prior to staining. Cells were washed with PBS, fixed with 4% paraformaldehyde, permeablized with 0.2% Triton X in PBS, blocked with 1% bovine serum albumin in PBS, and probed with monoclonal anti-CA antibodies. This was followed by detection with Alexa Fluor 488 anti-rat antibody (Molecular Probes, Inc). Slides were viewed using a Deltavison Olympus IX70 inverted microscope through a x100 objective lens with Softworx image acquisition software.
RESULTS
Determination of the amount of VLP required for abrogation. During retroviral maturation, PR cleaves the Gag precursor protein and assembly of mature cores follows (33). To test the possible involvement of these processes in allowing an interaction between CA and restriction factor, we planned to investigate the abrogation properties of a series of mutants defective for processing/maturation. However, before this we felt it important to properly characterize the quantity of wild-type CA needed for abrogation in the cell types under investigation. Human TE 671 cells (expressing Ref1 and therefore restricting N-tropic MLV), as well as murine BALB/3T3 (Fv1b, restricting mainly N-MLV) and NIH 3T3 (expressing Fv1n and restricting B-MLV) cells were pretreated with increasing amounts of restricted virus and then challenged with equal amounts of N-tropic and B-tropic virus carrying a green fluorescent protein vector. The percentage of cells transduced was determined by FACS analysis and plotted against the amount of CA used for abrogation (Fig. 1). Abrogating virus was added as virus-like particles (VLPs) produced by transient transfection of 293T cells with plasmids encoding Gag-Pol, vesicular stomatitis virus G protein protein, and a retroviral vector encoding LacZ.
Significant differences were seen between the cell lines. As expected, treatment of TE671 cells with N-tropic VLP had little or no effect on infectivity of B-tropic virus, but a steady increase in the titer of N-MLV was seen with increasing amounts of abrogating VLPs (Fig. 1A). It was possible to raise the infectivity of the N-tropic virus to a level that was almost equal to that of the B-tropic virus, implying that addition of VLPs containing 800 ng CA per well plated 24 h previously with 4 x 104 cells could approach saturation of Ref1 restriction. By contrast, it proved much more difficult to saturate Fv1n restriction in NIH-3T3 cells (Fig. 1B). Addition of VLPs containing B-tropic CA clearly reduced restriction of B-MLV while having little effect on N-MLV. However, even with the highest concentration of added VLPs, an appreciable difference in titers of N-MLV and B-MLV was still apparent. We attribute this to the relatively high levels of Fv1n present in NIH 3T3 cells (40). With BALB/3T3 cells, almost complete abrogation of Fv1b restriction was seen by N-tropic VLP (Fig. 1C). However, addition of N-tropic VLP also increased the apparent titer of B-tropic MLV. This is consistent with the previous observation of low-level restriction of B-MLV by Fv1b when overexpressed (2). The differences between the cell lines were highly reproducible, with virtually identical titration curves in three independent experiments; we therefore conclude that measuring abrogation responses following the addition of VLPs containing 800 ng CA per well to TE671 and BALB/3T3 cells and 1,700 ng to NIH 3T3 cells provides a good indication of the ability of CA to interact with Fv1 and Ref1.
Gag processing is required for abrogation. With a view to the examination of Gag processing/maturation requirements for the interaction of the virus with the restriction factor, a series of mutations, described in Table 1, were introduced by site-directed mutagenesis into the gag-pol constructs used to produce N- and B-tropic VLPs. This series comprised active site mutations in PR and RT, changes inhibiting MA-p12, p12-CA, and CA-NC processing, as well as a mutation preventing the formation of a salt bridge between P1 and D54 critical for the assembly of processed CA into mature cores (36).
To examine the patterns of protein expression in virions prepared using these plasmids, Western blot analysis of released virus concentrated by ultracentrifugation was performed, using anti-CA and anti-p12 antibodies. Results with N-tropic virions are shown in Fig. 2. B-tropic VLPs gave identical results (data not shown). On the whole, results were consistent with our expectations. Thus, after probing with anti-CA (Fig. 2A), we observed a band with the mobility of wild-type CA in RT–, MAxp12, and D54A. A band of the size predicted for unprocessed Gag was observed in PR–, and bands corresponding to p12-CA and CA-NC were seen in p12xCA and CAxNC. Somewhat surprisingly, there was a significant amount of apparently uncleaved Gag precursor in the RT– mutant. The results with anti-p12 (Fig. 2B) were slightly less straightforward and were complicated in part by the incomplete processing of MA-p12 seen in wild-type virus and the apparent nonreactivity of our anti-p12 sera with full-length Gag. However Fig. 2B does reveal bands of the expected size in MAxp12 (27 kDa) and p12xCA (42 kDa) and is consistent with reduced Gag processing in RT– (compare relative intensities of the 12- and 27-kDa bands in the wild type and RT–). The presence of a 27-kDa band in CAxNC suggests impaired cleavage of MA-p12 in this mutant. This was not reported in the initial study (23) describing the properties of Gag cleavage mutants; we have no explanation for this discrepancy but note that it was seen with both N- and B-tropic virions. D54A was examined on different gels; it showed processing identical to that with wild-type MLV (data not shown).
We next examined the abrogation properties of our panel of viruses. Cells were treated with VLPs containing restricted CA for 3 h and then infected with restricted virus. eGFP-positive cells were enumerated 3 days later. Results from such an experiment are shown in Fig. 3. With all three cell types, significant abrogation was seen only with wild-type, RT–, and MAxp12 CA. Abrogation was abolished, essentially completely, by the PR–, p12xCA, CAxNC, and D54A mutations. These findings imply that for abrogation to occur, complete processing at both the N and C termini of CA is necessary. Further, the absence of abrogation with D54A hints at a possible role for formation of an oligomeric condensed core in addition to processing.
Expression of processed CA in restricting cells does not abolish restriction. Gag processing occurs following virus release from the infected cell. To ask whether the processing requirement might explain the absence of abrogation in cells chronically infected with restricted virus, we wanted to determine whether the expression of processed CA, with an intact N-terminal proline residue, in cell lines showing Ref1 or Fv1 activity affected restriction. To achieve this aim, we prepared a Gag derivative, truncated after CA and containing the FMDV 2A protease between p12 and CA (Fig. 4A). Based on previous studies of autoproteolytic cleavage in FMDV (26, 27), expression and processing of this construct would be predicted to release the mature form of CA. NIH 3T3, BALB/3T3, M. dunni, and TE671 cells were transduced with vectors expressing these constructs (pLIB-BCA-puro or pLIB-NCA-puro), and single-cell clones were derived.
CA expression in cell lysates was examined by Western blot analysis (Fig. 4B). All cell lines showed the expected 30-kDa band when probed with an anti-CA monoclonal antibody. In several of the cell lines, unprocessed 60-kDa p15-p12-2A-CA was also observed.
Given the potential importance of CA N-terminal proline for the formation of a salt bridge with D54 and the requirement of this proposed salt bridge for abrogation as revealed by the D54A mutation, we thought it important to ensure that the N-terminal proline was still present after processing by the FMDV 2A protease. CA from an M. dunni NCA-expressing clone was immunoprecipitated, digested with Asp-N, and analyzed by mass spectrometry. The predicted PLRLGGNGQLQYWPFSSS N-terminal fragment with an m/z ratio of 2,007 as well as its expected tryptophan oxidation product (m/z 2,039) were identified (Fig. 4C), confirming precise processing by the FMDV 2A protease.
To test whether CA was expressed throughout the cell and available to interact with the restriction factor, M. dunni NCA cells, which show little or no unprocessed p60 (Fig. 4B), were examined by immunostaining for CA. Diffuse staining was observed in all cells but with some concentration in punctate structures (Fig. 5A). The pattern of free CA in these cells is strikingly different from that of unprocessed Gag present in chronically infected M. dunni cells producing N-tropic MLV (Fig. 5B), suggesting that upon processing, CA is freed from the perinuclear and membrane localizations imposed by the presence of MA and thus able to move freely in the cytoplasm, where it might interact with restriction factors. It can also enter the nucleus.
To determine the ability of CA-expressing cells to restrict MLV infection, these cells were challenged with equal amounts of N- and B-tropic virus carrying the pLNCG (eGFP) vector. The ratio between the percentages of cells transduced with N- and B-tropic virus was determined. A nonrestricting cell line would be transduced equally well by both the N- and the B-tropic viruses and so would give a ratio of 1. Cell lines restricting the N-tropic virus would give a ratio of >1, whereas cell lines restricting the B-tropic virus would give a ratio of <1.
Titers of infecting viruses were normalized with M. dunni cells that do not express an MLV restriction factor to give a ratio of 1 (Fig. 6). This ratio was not affected by the expression of either N or B CA in these cells. For the cell lines known to restrict N-tropic virus (BALB/3T3 and TE671), wild-type cells gave a ratio of >1. This was unaffected by the expression of N or B CA in these lines. The converse applied to NIH-3T3 cells that restrict B-tropic MLV. Wild-type cells gave the expected ratio of <1, with the presence of N or B CA having no effect. Identical results were obtained using a second set of independently derived clones. These data indicate that intracellular expression of fully processed CA is not sufficient to abrogate restriction.
DISCUSSION
The genetics of MLV restriction by Fv1 and Ref1, where single amino acid changes can alter host range (18), are most easily explained by specific interactions between a restriction factor and its viral target. Despite this, there are no reports documenting such an interaction directly. Our data help rationalize this absence. They show that mutations in the PR cleavage sites flanking CA result in the formation of VLPs that cannot abrogate the restriction factor. A requirement for Gag processing would explain why virus-producing cells still show complete Fv1 restriction (8). Further, the lack of abrogation seen with D54A and following expression of CA in mature form strongly suggests that in addition to processing, assembly of CA into cores is also required. We suggest that CA recognition by restriction factor occurs only in the context of intact viral cores. If CA polymerization occurs only in budded virions following appropriate proteolysis, the lack of interaction between CA and Fv1 observed in yeast two-hybrid systems (unpublished data) or between CA and Fv1/Ref1 in cells expressing mature CA is therefore readily explicable.
Aiken and colleagues have recently reported very similar studies of HIV-1 restriction by the Lv1 gene of owl monkeys (9). They show that abrogation requires Gag processing and that mutations affecting core stability influence abrogation properties and conclude that the target for restriction is a stable, polymeric capsid. One difference between their results and those reported here concerns the requirement for processing at the C-terminal side of CA. Our results indicate that cleavage between CA and NC is required for abrogation with MLV (Fig. 3A), but this does not seem to be the case with HIV-1 (9). This most likely reflects differences between HIV-1 and MLV in the process of core assembly; MLV CAxNC is reported not to form any cores (23), whereas the corresponding HIV-1 mutant does form a core, which, though morphologically immature, is stable and polymeric (9, 38).
Why should CA polymerization be required for restriction factor binding? Two alternative explanations might be advanced. First, following processing and/or assembly, structural arrangements occur within monomeric CA, forming or presenting a novel structure that can bind restriction factor. Although some rearrangement upon release of the N-terminal sequences allowing an interaction between P1 and D54 must occur during MLV maturation, comparison with pre- and postcleavage forms of human T-cell leukemia virus type 1 (6) suggests that any overall changes are likely to be small and not to involve the regions of CA involved in restriction factor binding (21, 31). We therefore favor the alternative hypothesis, namely that a binding site for restriction factor is created by the juxtaposition of CA monomers occurring during assembly. Further structural information will be required before we can distinguish between these alternatives.
What might be the effect of binding of restriction factor to incoming virions? It could be that binding itself can inhibit virus replication, perhaps by altering the stability of viral cores (10) and thereby influencing the process of reverse transcription. Alternatively, restriction factor binding might mediate some chemical modification of CA, for example, sumolation or ubiquitination, leading to an altered virion/preintegration complex fate (12). It is interesting that interaction with Ref1 (34) and Fv1 (16) results in restriction before and after reverse transcription, respectively, even though the abrogation requirements are identical. In particular, there is no need for active RT within the abrogating particles. Thus, despite very similar binding characteristics, Fv1 and Ref1 restrict retrovirus replication by different mechanisms.
ACKNOWLEDGMENTS
We thank S. Howell for assistance with mass spectrometry and G. Mortuza for providing protein samples.
This work was supported by the UK Medical Research Council.
M.P.D. and M.B. contributed equally to this study.
REFERENCES
Bieniasz, P. D. 2004. Intrinsic immunity: a front-line defense against viral attack. Nat. Immunol. 5:1109-1115.
Bock, M., K. N. Bishop, G. Towers, and J. P. Stoye. 2000. Use of a transient assay for studying the genetic determinants of Fv1 restriction. J. Virol. 74:7422-7430.
Boone, L. R., C. L. Innes, and C. K. Heitman. 1990. Abrogation of Fv-1 restriction by genome-deficient virions produced by a retrovirus packaging cell line. J. Virol. 64:3376-3381.
Chesebro, B., W. Britt, L. Evans, K. Wehrly, J. Nishio, and M. Cloyd. 1983. Characterization of monoclonal antibodies reactive with murine leukemia viruses: use in analysis of strains of friend MCF and Friend ecotropic murine leukemia virus. Virology 127:134-148.
Coligan, J. E. 1996. Current protocols in protein science. Wiley, Brooklyn, N.Y.
Cornilescu, C. C., F. Bouamr, X. Yao, C. Carter, and N. Tjandra. 2001. Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein. J. Mol. Biol. 306:783-797.
Duran-Troise, G., R. H. Bassin, A. Rein, and B. I. Gerwin. 1977. Loss of Fv-1 restriction in Balb/3T3 cells following infection with a single N tropic murine leukemia virus particle. Cell 10:479-488.
Duran-Troise, G., R. H. Bassin, B. F. Wallace, and A. Rein. 1981. Balb/3T3 cells chronically infected with N-tropic murine leukemia virus continue to express Fv-1b restriction. Virology 112:795-799.
Forshey, B. M., J. Shi, and C. Aiken. 2005. Structural requirements for recognition of the human immunodeficiency virus type 1 core during host restriction in owl monkey cells. J. Virol. 79:869-875.
Forshey, B. M., U. von Schwedler, W. I. Sundquist, and C. Aiken. 2002. Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J. Virol. 76:5667-5677.
Fu, W., and A. Rein. 1993. Maturation of dimeric viral RNA of Moloney murine leukemia virus. J. Virol. 67:5443-5449.
Goff, S. P. 2004. Retrovirus restriction factors. Mol. Cell 16:849-859.
Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Hartley, J. W., W. P. Rowe, and R. J. Huebner. 1970. Host-range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5:221-225.
Hatziioannou, T., D. Perez-Caballero, A. Yang, S. Cowan, and P. D. Bieniasz. 2004. Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc. Natl. Acad. Sci. USA 101:10774-10779.
Jolicoeur, P. 1979. The Fv-1 gene of the mouse and its control of murine leukemia virus replication. Curr. Top. Microbiol. Immunol. 86:67-122.
Keckesova, Z., L. M. Ylinen, and G. J. Towers. 2004. The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc. Natl. Acad. Sci. USA 101:10780-10785.
Kozak, C. A., and A. Chakraborti. 1996. Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. Virology 225:300-305.
Lilly, F. 1970. Fv-2: identification and location of a second gene governing the spleen focus response to Friend leukemia virus in mice. J. Natl. Cancer Inst. 45:163-169.
Lowe, D. M., V. Parmar, S. D. Kemp, and B. A. Larder. 1991. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 282:231-234.
Mortuza, G. B., L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye, and I. A. Taylor. 2004. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431:481-485.
Nisole, S., C. Lynch, J. P. Stoye, and M. W. Yap. 2004. A Trim5-cyclophilin A fusion protein found in owl monkey kidney cells can restrict HIV-1. Proc. Natl. Acad. Sci. USA 101:13324-13328.
Oshima, M., D. Muriaux, J. Mirro, K. Nagashima, K. Dryden, M. Yeager, and A. Rein. 2004. Effects of blocking individual maturation cleavages in murine leukemia virus Gag. J. Virol. 78:1411-1420.
Perkins, D. N., D. J. Pappin, D. M. Creasy, and J. S. Cottrell. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551-3567.
Pietschmann, T., M. Heinkelein, M. Heldmann, H. Zentgraf, A. Rethwilm, and D. Lindemann. 1999. Foamy virus capsids require the cognate envelope protein for particle export. J. Virol. 73:2613-2621.
Ryan, M. D., and J. Drew. 1994. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13:928-933.
Ryan, M. D., A. M. King, and G. P. Thomas. 1991. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen. Virol. 72:2727-2732.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Sayah, D. M., E. Sokolskaja, L. Berthoux, and J. Luban. 2004. Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1. Nature 430:569-573.
Soneoka, Y., P. M. Cannon, E. E. Ramsdale, J. C. Griffiths, G. Romano, S. M. Kingsman, and A. J. Kingsman. 1995. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23:628-633.
Stevens, A., M. Bock, S. Ellis, P. LeTissier, K. N. Bishop, M. W. Yap, W. Taylor, and J. P. Stoye. 2004. Retroviral capsid determinants of Fv1 NB and NR tropism. J. Virol. 78:9592-9598.
Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-853.
Swanstrom, R., and J. W. Wills. 1997. Synthesis, assembly, and processing of viral proteins, p. 263-334. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Towers, G., M. Bock, S. Martin, Y. Takeuchi, J. P. Stoye, and O. Danos. 2000. A conserved mechanism of retrovirus restriction in mammals. Proc. Natl. Acad. Sci. USA 97:12295-12299.
Towers, G., M. Collins, and Y. Takeuchi. 2002. Abrogation of Ref1 retrovirus restriction in human cells. J. Virol. 76:2548-2550.
von Schwedler, U. K., T. L. Stemmler, V. Y. Klishko, S. Li, K. H. Albertine, D. R. Davis, and W. I. Sundquist. 1998. Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J. 17:1555-1568.
Vorm, O., and P. Roepstorff. 1994. Peptide sequence information derived by partial acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry. Biol. Mass Spectrom. 23:734-740.
Wiegers, K., G. Rutter, H. Kottler, U. Tessmer, H. Hohenberg, and H. G. Krausslich. 1998. Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites. J. Virol. 72:2846-2854.
Yap, M. W., S. Nisole, C. Lynch, and J. P. Stoye. 2004. Trim5alpha protein restricts both HIV-1 and murine leukemia virus. Proc. Natl. Acad. Sci. USA 101:10786-10791.
Yap, M. W., and J. P. Stoye. 2003. Intracellular localisation of Fv1. Virology 307:76-89.(Mark P. Dodding, Michael )
ABSTRACT
Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.
INTRODUCTION
It is becoming increasingly clear that mammalian cells can express a variety of factors that limit retroviral replication. One class of these factors targets the retroviral capsid (CA) protein and blocks replication at a stage postentry but prior to integration of the viral genetic material into the cell genome. Members of this family are known as restriction factors (1, 12). The best-characterized member of this family is the mouse Fv1 (19) gene, which determines susceptibility to infection by murine leukemia virus (MLV). Fv1 has two major alleles, known as Fv1n and Fv1b, whose restriction characteristics determine the host range of different MLV strains, allowing the division of MLV into a number of classes, two of which are known as N tropic and B tropic (16). An Fv1n/n mouse (or cell line) restricts the B-tropic virus but is permissive to N-tropic MLV. An Fv1b/b mouse restricts the N-tropic virus but not B-tropic MLV. Resistance is semidominant; heterozygous animals restrict both classes of virus. NB-tropic viruses are relatively insensitive to Fv1 restriction (14). A similar form of MLV restriction exists in human cell lines. Restriction is comparable to that of the B allele of Fv1 in that the N-tropic virus is restricted while the B-tropic virus is not. This restriction factor has been named Ref1 (34). Two members of the TRIM family of proteins, Trim5 and Trim1, have been implicated as components of this restriction (15, 17, 39). Trim5 from several primate species can also restrict HIV-1 (22, 29, 32).
The major determinant of MLV restriction has been mapped to the CA protein. A single amino acid change within the capsid (R110E) will convert an N-tropic virus to a B-tropic virus and a Ref1-sensitive MLV to a Ref1-resistant virus (18, 34). Despite this, it has so far been impossible to show a direct interaction between CA and Fv1 using standard methods, such as yeast two-hybrid or coimmunoprecipitation techniques (unpublished data). However, restriction by Ref1 or Fv1 can be overcome when infecting cells at a high multiplicity of infection (7, 35). Further, pretreatment of cells with restricted virus allows unrestricted infection by a second, challenge virus (3). This suggests that restriction factor is limiting and can be saturated, or "abrogated," by restricted virus. Consistent with this proposition, natural levels of Fv1 expression appear very low (40). Abrogation therefore provides the basis of a method for studying the interaction between CA and restriction factors.
Despite the fact that the restriction factor can be abrogated by exposure to restricted virus at a high multiplicity of infection, chronically infected cell lines in which high levels of restricted Gag polyprotein are expressed can still restrict incoming virus (8, 16). Two explanations, not necessarily mutually exclusive, might be advanced to explain this apparent paradox. First, Gag processing and/or assembly might be required for CA recognition by Fv1. Only after a virus is released from the producer cell is the viral Gag cleaved into its four constituent parts—matrix (MA), p12, CA, and nucleocapsid (NC)—by the viral protease (PR), thereby leading to the condensation of the CA component to form a spherical core containing the RNA genome. It does not seem unreasonable to suppose that Fv1 interacts only with the mature, multimerized form of CA. Alternatively, it seems possible that the intracellular localization of CA during viral egress and ingress plays a critical role. Active Fv1 is found in association with tubules of the trans-Golgi network (40), and it may be that Gag polyprotein never meets Fv1 during its outgoing transit through the cell. In this report we describe experiments designed to address these possibilities, focusing on establishing the relationship between Gag processing and abrogation.
MATERIALS AND METHODS
Cell culture and virus preparations. 293T, Mus dunni, BALB/3T3, NIH 3T3, and TE671 cell lines were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and penicillin/streptomycin (Sigma). Viruses were prepared by transient transfection of 293T cells using the Promega Profection mammalian transfection system, typically with 7 μg gag-pol expression vector, pCIG3 N, B, pHIT 60 (30), or derivatives described below; 7 μg of vesicular stomatitis virus G protein expression vector, pczVSV-G (25), and 7 μg of either pLNCG (encoding enhanced green fluorescent protein [eGFP]) (39) or pHIT111 (lacZ) (30) or pLIB-NCA/BCA-puro (see below).
Generation of mutant gag-pol expression vectors. Mutants of pCIG3 N and B plasmids were made by PCR site-directed mutagenesis using PfuTurbo DNA polymerase (Stratagene) according to the manufacturer's protocol with the following oligonucleotides: protease D32L (PR–), forward, 5'-CCGTCACCTTCCTGGTGCTTACTGGGGCCCAACACTCC-3', and reverse, 5'-GGAGTGTTGGGCCCCAGTAAGCACCAGGAAGGTGACGGG-3'; reverse transcriptase D224E (RT–), forward, 5'-CCTGCTACAGTACGTGGAGGACATACTACTGGCC-3', and reverse, 5'-GGCCAGTAGTATGTCCTCCACGTACTGTAGCAGG-3'; MA-p12 (MAxp12), forward, 5'-CCCCGATCTGCCCTTGATCCTGCTCTTACCCC-3', and reverse, 5'-GGGGTAAGAGCAGGATCAAGGGCAGATCGGGG-3'; p12-CA (p12xCA), forward, 5'-CCACCTCTCGGGCTGACCCACTCCGTTTGGGG-3', and reverse, 5'-CCCCAAACGGAGTGGGTCAGCCCGAGAGGTGG-3'; CA-NC (CAxNC), forward, 5'-GGAATGAGCAAACTTAGGGCCACCGTAGTTAG-3', and reverse, 5'-CTAACTACGGTGGCCCTAAGTTTGCTCATTTC-3'; CA D54A (D54A), forward, 5'-CAGCCCACCTGGGATGCCTGCCAGCAATTATTAG-3', and reverse, 5'-CTAATAATTGCTGGCAGGCATCCCAGGTGGGCTG-3'.
The sequences of the mutations introduced were verified by DNA sequencing.
Abrogation experiments. In all cases, cells were plated at a density of 4 x 104 per well in a 12-well plate 24 h prior to transduction. These were pretreated with 500 μl of the appropriate virus-like particles (VLP), at concentrations given below, for 3 h, before challenge with a fixed amount of either N- or B-tropic virus carrying the pLNCG (eGFP) vector. The percentage of transduced cells was analyzed by fluorescence-activated cell sorter (FACS) analysis after 3 days.
Derivation of single-cell clones expressing CA. To insert a foot-and-mouth disease virus (FMDV) 2A protease between p12 and CA, thereby allowing intracellular expression of properly processed CA, the gag region of pCIG3 N and B was amplified by PCR in two sections. Primers were designed to produce an EcoRI-MA-p12-(N-terminal 2A)-HindIII PCR product and a HindIII-(C-terminal 2A)-CA-MfeI PCR product. The following primer sets were used: for MA-p12-2A, forward, 5'-GCGAATTCACATGGGACAGACCGTAACC-3', and reverse, 5'-GCAAGCTTAAGAAGGTCAAAATTCAACAGAAAGCCCCGAGAGGTGGTGG-3'; 2A-CA, forward, 5'-GCAAGCTTGCGGGAGACGTCGAGTCCAACCCCGGGCCACTCCGTTTG-3', and reverse, 5'-CGCAATTGTTACAAAAGTTTGCTCATTTCTCTA-3'. Each product was cloned into pCRII-TOPO (Invitrogen). These plasmids were then digested with either EcoRI/HindIII for the MA-p12-2A insert or HindIII/MfeI for the 2A-CA insert. A triple ligation was performed with these two inserts and the pIRESpuro vector (Clontech) (previously cut with EcoRI), creating MA-p12-2A-CA-IRES-puro. The full construct was excised with EcoRI and StuI and ligated into pLIB (Clontech), previously cut with the same enzymes, producing pLIB-NCA-puro and pLIB-BCA-puro. Viral vectors carrying pLIB-NCA-puro and pLIB-BCA-puro were prepared following transfection of 293T cells. Serial dilutions of each virus were prepared (10–1, 10–2, 10–3, and 10–4), with 100 μl of each used to infect a 6-cm plate with 5 x 105 NIH 3T3, BALB/3T3, M. dunni, or TE671 cells. After 2 days, puromycin was added to the media at a concentration of 2 μg/ml.
Two weeks later, plates with separate colonies could be identified for each cell line and expression construct. Three colonies of each type were isolated with cloning rings (Sigma) and grown as individual cultures. Cells were screened for CA expression by Western blot analysis.
Western blotting. Virions were pelleted by centrifugation at 55,000 rpm for 90 min in a Beckman TLA 120.1 rotor, washed once with phosphate-buffered saline (PBS), suspended in sodium dodecyl sulfate (SDS)/loading dye containing 10 mM dithiothreitol (DTT), and heated to 90°C for 5 min before electrophoresis on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. Proteins were transferred to polyvinylidene difluoride membranes (Millipore) and probed with monoclonal anti-CA (4) or polyclonal anti-p12 antibodies. Cell lysates from N-tropic CA/B-tropic CA (NCA/BCA)-expressing single-cell clones were prepared by adding 500 μl triple detergent lysis buffer (28) to confluent cells in a 6-cm dish and cleared by centrifugation. Following the addition of SDS/loading dye containing 10 mM DTT to the supernatant, samples were heated to 90°C for 5 min, and 35 μl was loaded onto a 12% SDS-PAGE gel and processed as described above.
CA enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G fractions from goat anti-CA polyclonal antisera (Viromed) were purified on a protein A column (13). This material was biotinylated using the Immunoprobe Biotinylation kit (Sigma) according to the manufacturer's guidelines. Wells from a 96-well Reacti-Bind streptavidin plate (Pierce) were washed three times with 250 μl PBS, 0.1% bovine serum albumin, 0.05% Tween before being incubated overnight at 4°C with 100 μl of 25 μg/ml biotinylated anti-CA (diluted in wash buffer). Plates were washed three more times before the addition of 100 μl of viral sample, pretreated with 1% NP-40 for 15 min at 37°C to disrupt viral membranes, and then incubated at room temperature for 1 h. After three further washes, rat monoclonal anti-CA antibody (100 μl) was then added at a concentration of 10 μg/ml (diluted in wash buffer), and the plates were incubated for a further 90 min at room temperature. After three more washes, 100 μl of monoclonal goat anti-rat immunoglobulin G conjugated to calf intestinal phosphatase (Pierce) was added at a concentration of 6 μg/ml and incubated at room temperature for 1 h. This was then washed 10 times with wash buffer. Binding of this antibody was detected using the Phosphatase Substrate kit (Pierce) according to the manufacturer's protocol. A405 was measured using a Thermo Labsystems Multiskan Ascent spectrophotometer. Concentrations were determined from the mean A405 of three replicates. Standard curves were obtained with N-terminally His-tagged CA purified from Escherichia coli (21).
Immunoprecipitation and fingerprinting by matrix-assisted laser desorption ionization mass spectrometry. M. dunni cells expressing CA from N-tropic MLV were lysed using a nondenaturing lysis buffer, and the CA was immunoprecipitated using rat monoclonal anti-pCA antibody bound to protein G beads (5). Beads were then resuspended in SDS/loading dye containing 10 mM DTT and then incubated at 90°C for 5 min to detach the bound protein. Following centrifugation, the supernatant was loaded onto a 12% SDS-PAGE gel. After electrophoresis, gels were stained with Simply Blue Safe Stain colloidal Coomassie (Invitrogen), and the band at 30 kDa was excised, reduced with dithiothreitol, and alkylated using iodoacetamide. Gel slices were dried and reswollen in a sufficient volume to cover 4 ng/μl endoproteinase Asp-N (sequencing grade; Roche) in 5 mM ammonium bicarbonate. After overnight digestion at 32°C, the supernatant was acidified by the addition of a 1/10 volume of 4% trifluoroacetic acid. Peptide mass fingerprinting was performed using a Reflex III matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), equipped with a nitrogen laser and a Scout-384 probe, to obtain positive ion mass spectra of digested protein with pulsed ion extraction in reflectron mode. An accelerating voltage of 26 kV was used with detector bias gating set to 2 kV and a mass cutoff of m/z 650. Matrix surfaces were prepared using recrystallized -cyano-4-hydroxycinammic acid and nitrocellulose, using the fast evaporation method (37) Digestion supernatant (0.4 μl) was deposited on the matrix surface and allowed to dry prior to desalting with water. Peptide mass fingerprints were compared to the nonredundant protein database placed in the public domain by the National Center for Biotechnology Information using the program MASCOT (24).
Immunofluorescence. Cells were plated at a density of 2 x 104 per well on a glass coverslip in a 12-well plate 24 h prior to staining. Cells were washed with PBS, fixed with 4% paraformaldehyde, permeablized with 0.2% Triton X in PBS, blocked with 1% bovine serum albumin in PBS, and probed with monoclonal anti-CA antibodies. This was followed by detection with Alexa Fluor 488 anti-rat antibody (Molecular Probes, Inc). Slides were viewed using a Deltavison Olympus IX70 inverted microscope through a x100 objective lens with Softworx image acquisition software.
RESULTS
Determination of the amount of VLP required for abrogation. During retroviral maturation, PR cleaves the Gag precursor protein and assembly of mature cores follows (33). To test the possible involvement of these processes in allowing an interaction between CA and restriction factor, we planned to investigate the abrogation properties of a series of mutants defective for processing/maturation. However, before this we felt it important to properly characterize the quantity of wild-type CA needed for abrogation in the cell types under investigation. Human TE 671 cells (expressing Ref1 and therefore restricting N-tropic MLV), as well as murine BALB/3T3 (Fv1b, restricting mainly N-MLV) and NIH 3T3 (expressing Fv1n and restricting B-MLV) cells were pretreated with increasing amounts of restricted virus and then challenged with equal amounts of N-tropic and B-tropic virus carrying a green fluorescent protein vector. The percentage of cells transduced was determined by FACS analysis and plotted against the amount of CA used for abrogation (Fig. 1). Abrogating virus was added as virus-like particles (VLPs) produced by transient transfection of 293T cells with plasmids encoding Gag-Pol, vesicular stomatitis virus G protein protein, and a retroviral vector encoding LacZ.
Significant differences were seen between the cell lines. As expected, treatment of TE671 cells with N-tropic VLP had little or no effect on infectivity of B-tropic virus, but a steady increase in the titer of N-MLV was seen with increasing amounts of abrogating VLPs (Fig. 1A). It was possible to raise the infectivity of the N-tropic virus to a level that was almost equal to that of the B-tropic virus, implying that addition of VLPs containing 800 ng CA per well plated 24 h previously with 4 x 104 cells could approach saturation of Ref1 restriction. By contrast, it proved much more difficult to saturate Fv1n restriction in NIH-3T3 cells (Fig. 1B). Addition of VLPs containing B-tropic CA clearly reduced restriction of B-MLV while having little effect on N-MLV. However, even with the highest concentration of added VLPs, an appreciable difference in titers of N-MLV and B-MLV was still apparent. We attribute this to the relatively high levels of Fv1n present in NIH 3T3 cells (40). With BALB/3T3 cells, almost complete abrogation of Fv1b restriction was seen by N-tropic VLP (Fig. 1C). However, addition of N-tropic VLP also increased the apparent titer of B-tropic MLV. This is consistent with the previous observation of low-level restriction of B-MLV by Fv1b when overexpressed (2). The differences between the cell lines were highly reproducible, with virtually identical titration curves in three independent experiments; we therefore conclude that measuring abrogation responses following the addition of VLPs containing 800 ng CA per well to TE671 and BALB/3T3 cells and 1,700 ng to NIH 3T3 cells provides a good indication of the ability of CA to interact with Fv1 and Ref1.
Gag processing is required for abrogation. With a view to the examination of Gag processing/maturation requirements for the interaction of the virus with the restriction factor, a series of mutations, described in Table 1, were introduced by site-directed mutagenesis into the gag-pol constructs used to produce N- and B-tropic VLPs. This series comprised active site mutations in PR and RT, changes inhibiting MA-p12, p12-CA, and CA-NC processing, as well as a mutation preventing the formation of a salt bridge between P1 and D54 critical for the assembly of processed CA into mature cores (36).
To examine the patterns of protein expression in virions prepared using these plasmids, Western blot analysis of released virus concentrated by ultracentrifugation was performed, using anti-CA and anti-p12 antibodies. Results with N-tropic virions are shown in Fig. 2. B-tropic VLPs gave identical results (data not shown). On the whole, results were consistent with our expectations. Thus, after probing with anti-CA (Fig. 2A), we observed a band with the mobility of wild-type CA in RT–, MAxp12, and D54A. A band of the size predicted for unprocessed Gag was observed in PR–, and bands corresponding to p12-CA and CA-NC were seen in p12xCA and CAxNC. Somewhat surprisingly, there was a significant amount of apparently uncleaved Gag precursor in the RT– mutant. The results with anti-p12 (Fig. 2B) were slightly less straightforward and were complicated in part by the incomplete processing of MA-p12 seen in wild-type virus and the apparent nonreactivity of our anti-p12 sera with full-length Gag. However Fig. 2B does reveal bands of the expected size in MAxp12 (27 kDa) and p12xCA (42 kDa) and is consistent with reduced Gag processing in RT– (compare relative intensities of the 12- and 27-kDa bands in the wild type and RT–). The presence of a 27-kDa band in CAxNC suggests impaired cleavage of MA-p12 in this mutant. This was not reported in the initial study (23) describing the properties of Gag cleavage mutants; we have no explanation for this discrepancy but note that it was seen with both N- and B-tropic virions. D54A was examined on different gels; it showed processing identical to that with wild-type MLV (data not shown).
We next examined the abrogation properties of our panel of viruses. Cells were treated with VLPs containing restricted CA for 3 h and then infected with restricted virus. eGFP-positive cells were enumerated 3 days later. Results from such an experiment are shown in Fig. 3. With all three cell types, significant abrogation was seen only with wild-type, RT–, and MAxp12 CA. Abrogation was abolished, essentially completely, by the PR–, p12xCA, CAxNC, and D54A mutations. These findings imply that for abrogation to occur, complete processing at both the N and C termini of CA is necessary. Further, the absence of abrogation with D54A hints at a possible role for formation of an oligomeric condensed core in addition to processing.
Expression of processed CA in restricting cells does not abolish restriction. Gag processing occurs following virus release from the infected cell. To ask whether the processing requirement might explain the absence of abrogation in cells chronically infected with restricted virus, we wanted to determine whether the expression of processed CA, with an intact N-terminal proline residue, in cell lines showing Ref1 or Fv1 activity affected restriction. To achieve this aim, we prepared a Gag derivative, truncated after CA and containing the FMDV 2A protease between p12 and CA (Fig. 4A). Based on previous studies of autoproteolytic cleavage in FMDV (26, 27), expression and processing of this construct would be predicted to release the mature form of CA. NIH 3T3, BALB/3T3, M. dunni, and TE671 cells were transduced with vectors expressing these constructs (pLIB-BCA-puro or pLIB-NCA-puro), and single-cell clones were derived.
CA expression in cell lysates was examined by Western blot analysis (Fig. 4B). All cell lines showed the expected 30-kDa band when probed with an anti-CA monoclonal antibody. In several of the cell lines, unprocessed 60-kDa p15-p12-2A-CA was also observed.
Given the potential importance of CA N-terminal proline for the formation of a salt bridge with D54 and the requirement of this proposed salt bridge for abrogation as revealed by the D54A mutation, we thought it important to ensure that the N-terminal proline was still present after processing by the FMDV 2A protease. CA from an M. dunni NCA-expressing clone was immunoprecipitated, digested with Asp-N, and analyzed by mass spectrometry. The predicted PLRLGGNGQLQYWPFSSS N-terminal fragment with an m/z ratio of 2,007 as well as its expected tryptophan oxidation product (m/z 2,039) were identified (Fig. 4C), confirming precise processing by the FMDV 2A protease.
To test whether CA was expressed throughout the cell and available to interact with the restriction factor, M. dunni NCA cells, which show little or no unprocessed p60 (Fig. 4B), were examined by immunostaining for CA. Diffuse staining was observed in all cells but with some concentration in punctate structures (Fig. 5A). The pattern of free CA in these cells is strikingly different from that of unprocessed Gag present in chronically infected M. dunni cells producing N-tropic MLV (Fig. 5B), suggesting that upon processing, CA is freed from the perinuclear and membrane localizations imposed by the presence of MA and thus able to move freely in the cytoplasm, where it might interact with restriction factors. It can also enter the nucleus.
To determine the ability of CA-expressing cells to restrict MLV infection, these cells were challenged with equal amounts of N- and B-tropic virus carrying the pLNCG (eGFP) vector. The ratio between the percentages of cells transduced with N- and B-tropic virus was determined. A nonrestricting cell line would be transduced equally well by both the N- and the B-tropic viruses and so would give a ratio of 1. Cell lines restricting the N-tropic virus would give a ratio of >1, whereas cell lines restricting the B-tropic virus would give a ratio of <1.
Titers of infecting viruses were normalized with M. dunni cells that do not express an MLV restriction factor to give a ratio of 1 (Fig. 6). This ratio was not affected by the expression of either N or B CA in these cells. For the cell lines known to restrict N-tropic virus (BALB/3T3 and TE671), wild-type cells gave a ratio of >1. This was unaffected by the expression of N or B CA in these lines. The converse applied to NIH-3T3 cells that restrict B-tropic MLV. Wild-type cells gave the expected ratio of <1, with the presence of N or B CA having no effect. Identical results were obtained using a second set of independently derived clones. These data indicate that intracellular expression of fully processed CA is not sufficient to abrogate restriction.
DISCUSSION
The genetics of MLV restriction by Fv1 and Ref1, where single amino acid changes can alter host range (18), are most easily explained by specific interactions between a restriction factor and its viral target. Despite this, there are no reports documenting such an interaction directly. Our data help rationalize this absence. They show that mutations in the PR cleavage sites flanking CA result in the formation of VLPs that cannot abrogate the restriction factor. A requirement for Gag processing would explain why virus-producing cells still show complete Fv1 restriction (8). Further, the lack of abrogation seen with D54A and following expression of CA in mature form strongly suggests that in addition to processing, assembly of CA into cores is also required. We suggest that CA recognition by restriction factor occurs only in the context of intact viral cores. If CA polymerization occurs only in budded virions following appropriate proteolysis, the lack of interaction between CA and Fv1 observed in yeast two-hybrid systems (unpublished data) or between CA and Fv1/Ref1 in cells expressing mature CA is therefore readily explicable.
Aiken and colleagues have recently reported very similar studies of HIV-1 restriction by the Lv1 gene of owl monkeys (9). They show that abrogation requires Gag processing and that mutations affecting core stability influence abrogation properties and conclude that the target for restriction is a stable, polymeric capsid. One difference between their results and those reported here concerns the requirement for processing at the C-terminal side of CA. Our results indicate that cleavage between CA and NC is required for abrogation with MLV (Fig. 3A), but this does not seem to be the case with HIV-1 (9). This most likely reflects differences between HIV-1 and MLV in the process of core assembly; MLV CAxNC is reported not to form any cores (23), whereas the corresponding HIV-1 mutant does form a core, which, though morphologically immature, is stable and polymeric (9, 38).
Why should CA polymerization be required for restriction factor binding? Two alternative explanations might be advanced. First, following processing and/or assembly, structural arrangements occur within monomeric CA, forming or presenting a novel structure that can bind restriction factor. Although some rearrangement upon release of the N-terminal sequences allowing an interaction between P1 and D54 must occur during MLV maturation, comparison with pre- and postcleavage forms of human T-cell leukemia virus type 1 (6) suggests that any overall changes are likely to be small and not to involve the regions of CA involved in restriction factor binding (21, 31). We therefore favor the alternative hypothesis, namely that a binding site for restriction factor is created by the juxtaposition of CA monomers occurring during assembly. Further structural information will be required before we can distinguish between these alternatives.
What might be the effect of binding of restriction factor to incoming virions? It could be that binding itself can inhibit virus replication, perhaps by altering the stability of viral cores (10) and thereby influencing the process of reverse transcription. Alternatively, restriction factor binding might mediate some chemical modification of CA, for example, sumolation or ubiquitination, leading to an altered virion/preintegration complex fate (12). It is interesting that interaction with Ref1 (34) and Fv1 (16) results in restriction before and after reverse transcription, respectively, even though the abrogation requirements are identical. In particular, there is no need for active RT within the abrogating particles. Thus, despite very similar binding characteristics, Fv1 and Ref1 restrict retrovirus replication by different mechanisms.
ACKNOWLEDGMENTS
We thank S. Howell for assistance with mass spectrometry and G. Mortuza for providing protein samples.
This work was supported by the UK Medical Research Council.
M.P.D. and M.B. contributed equally to this study.
REFERENCES
Bieniasz, P. D. 2004. Intrinsic immunity: a front-line defense against viral attack. Nat. Immunol. 5:1109-1115.
Bock, M., K. N. Bishop, G. Towers, and J. P. Stoye. 2000. Use of a transient assay for studying the genetic determinants of Fv1 restriction. J. Virol. 74:7422-7430.
Boone, L. R., C. L. Innes, and C. K. Heitman. 1990. Abrogation of Fv-1 restriction by genome-deficient virions produced by a retrovirus packaging cell line. J. Virol. 64:3376-3381.
Chesebro, B., W. Britt, L. Evans, K. Wehrly, J. Nishio, and M. Cloyd. 1983. Characterization of monoclonal antibodies reactive with murine leukemia viruses: use in analysis of strains of friend MCF and Friend ecotropic murine leukemia virus. Virology 127:134-148.
Coligan, J. E. 1996. Current protocols in protein science. Wiley, Brooklyn, N.Y.
Cornilescu, C. C., F. Bouamr, X. Yao, C. Carter, and N. Tjandra. 2001. Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein. J. Mol. Biol. 306:783-797.
Duran-Troise, G., R. H. Bassin, A. Rein, and B. I. Gerwin. 1977. Loss of Fv-1 restriction in Balb/3T3 cells following infection with a single N tropic murine leukemia virus particle. Cell 10:479-488.
Duran-Troise, G., R. H. Bassin, B. F. Wallace, and A. Rein. 1981. Balb/3T3 cells chronically infected with N-tropic murine leukemia virus continue to express Fv-1b restriction. Virology 112:795-799.
Forshey, B. M., J. Shi, and C. Aiken. 2005. Structural requirements for recognition of the human immunodeficiency virus type 1 core during host restriction in owl monkey cells. J. Virol. 79:869-875.
Forshey, B. M., U. von Schwedler, W. I. Sundquist, and C. Aiken. 2002. Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J. Virol. 76:5667-5677.
Fu, W., and A. Rein. 1993. Maturation of dimeric viral RNA of Moloney murine leukemia virus. J. Virol. 67:5443-5449.
Goff, S. P. 2004. Retrovirus restriction factors. Mol. Cell 16:849-859.
Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Hartley, J. W., W. P. Rowe, and R. J. Huebner. 1970. Host-range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Virol. 5:221-225.
Hatziioannou, T., D. Perez-Caballero, A. Yang, S. Cowan, and P. D. Bieniasz. 2004. Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc. Natl. Acad. Sci. USA 101:10774-10779.
Jolicoeur, P. 1979. The Fv-1 gene of the mouse and its control of murine leukemia virus replication. Curr. Top. Microbiol. Immunol. 86:67-122.
Keckesova, Z., L. M. Ylinen, and G. J. Towers. 2004. The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc. Natl. Acad. Sci. USA 101:10780-10785.
Kozak, C. A., and A. Chakraborti. 1996. Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. Virology 225:300-305.
Lilly, F. 1970. Fv-2: identification and location of a second gene governing the spleen focus response to Friend leukemia virus in mice. J. Natl. Cancer Inst. 45:163-169.
Lowe, D. M., V. Parmar, S. D. Kemp, and B. A. Larder. 1991. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 282:231-234.
Mortuza, G. B., L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye, and I. A. Taylor. 2004. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431:481-485.
Nisole, S., C. Lynch, J. P. Stoye, and M. W. Yap. 2004. A Trim5-cyclophilin A fusion protein found in owl monkey kidney cells can restrict HIV-1. Proc. Natl. Acad. Sci. USA 101:13324-13328.
Oshima, M., D. Muriaux, J. Mirro, K. Nagashima, K. Dryden, M. Yeager, and A. Rein. 2004. Effects of blocking individual maturation cleavages in murine leukemia virus Gag. J. Virol. 78:1411-1420.
Perkins, D. N., D. J. Pappin, D. M. Creasy, and J. S. Cottrell. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551-3567.
Pietschmann, T., M. Heinkelein, M. Heldmann, H. Zentgraf, A. Rethwilm, and D. Lindemann. 1999. Foamy virus capsids require the cognate envelope protein for particle export. J. Virol. 73:2613-2621.
Ryan, M. D., and J. Drew. 1994. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13:928-933.
Ryan, M. D., A. M. King, and G. P. Thomas. 1991. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen. Virol. 72:2727-2732.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Sayah, D. M., E. Sokolskaja, L. Berthoux, and J. Luban. 2004. Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1. Nature 430:569-573.
Soneoka, Y., P. M. Cannon, E. E. Ramsdale, J. C. Griffiths, G. Romano, S. M. Kingsman, and A. J. Kingsman. 1995. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23:628-633.
Stevens, A., M. Bock, S. Ellis, P. LeTissier, K. N. Bishop, M. W. Yap, W. Taylor, and J. P. Stoye. 2004. Retroviral capsid determinants of Fv1 NB and NR tropism. J. Virol. 78:9592-9598.
Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-853.
Swanstrom, R., and J. W. Wills. 1997. Synthesis, assembly, and processing of viral proteins, p. 263-334. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Towers, G., M. Bock, S. Martin, Y. Takeuchi, J. P. Stoye, and O. Danos. 2000. A conserved mechanism of retrovirus restriction in mammals. Proc. Natl. Acad. Sci. USA 97:12295-12299.
Towers, G., M. Collins, and Y. Takeuchi. 2002. Abrogation of Ref1 retrovirus restriction in human cells. J. Virol. 76:2548-2550.
von Schwedler, U. K., T. L. Stemmler, V. Y. Klishko, S. Li, K. H. Albertine, D. R. Davis, and W. I. Sundquist. 1998. Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J. 17:1555-1568.
Vorm, O., and P. Roepstorff. 1994. Peptide sequence information derived by partial acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry. Biol. Mass Spectrom. 23:734-740.
Wiegers, K., G. Rutter, H. Kottler, U. Tessmer, H. Hohenberg, and H. G. Krausslich. 1998. Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites. J. Virol. 72:2846-2854.
Yap, M. W., S. Nisole, C. Lynch, and J. P. Stoye. 2004. Trim5alpha protein restricts both HIV-1 and murine leukemia virus. Proc. Natl. Acad. Sci. USA 101:10786-10791.
Yap, M. W., and J. P. Stoye. 2003. Intracellular localisation of Fv1. Virology 307:76-89.(Mark P. Dodding, Michael )