2006, 80(1):342. DOI: 10.1128/JVI.80.1.342-352.2006.J. Virol. P. GoffA. Perrone, Kenia de los Santos, Szy-yuan Pu and Stephen Andrew Yueh, Juliana Leung, Subarna Bhattacharyya, Lucy InfectionSUMO-1 Addition Required Early in

MediatesVirus Capsid with Ubc9 and PIASy Interaction of Moloney Murine Leukemia

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JOURNAL OF VIROLOGY, Jan. 2006, p. 342–352 Vol. 80, No. 10022-538X/06/$08.00�0 doi:10.1128/JVI.80.1.342–352.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 andPIASy Mediates SUMO-1 Addition Required Early in Infection

Andrew Yueh,1† Juliana Leung,2 Subarna Bhattacharyya,1‡ Lucy A. Perrone,1§Kenia de los Santos,1 Szy-yuan Pu,3 and Stephen P. Goff1*

Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons,Columbia University, New York, New York 100321; Integrated Program in Cellular, Molecular, and Biophysical Studies,

College of Physicians and Surgeons, Columbia University, New York, New York 100322; and National HealthResearch Institutes, Division of Biotechnology and Pharmaceutical Research, Taipai, Taiwan, Republic of China3

Received 12 May 2005/Accepted 2 October 2005

Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier(SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leuke-mia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to aconsensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped toresidues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines nearthe Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation.Introduction of these mutations into the complete viral genome blocked virus replication. The mutantsexhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutantviruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but wereunable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The resultssuggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for earlyevents of infection, after reverse transcription and before nuclear entry and viral DNA integration.

The early events of the retroviral life cycle, especially theintracellular trafficking of the virus, are poorly understood (seereferences 11, 14, 18, and 51 for reviews). Soon after entry intothe cytoplasm of an infected cell, the viral RNA genome isreverse transcribed to give rise to a linear double-strandedDNA. This DNA is present in a large structure termed thepreintegration complex (PIC): the composition and structureof the PIC are poorly characterized, but it is likely to containGag proteins, reverse transcriptase (RT), integrase (IN), andseveral host proteins. There is good evidence that the Gagproteins, and especially capsid (CA), are important early ininfection. The PIC of the Moloney murine leukemia virus(Mo-MuLV) notably retains CA for many hours (5, 15). Thepresence of CA in the PIC is consistent with the effects of manygag mutations in the CA domain on early stages of infection (1,2, 30). CA is even more strongly implicated in early events ofinfection by its role as a target of the Fv1 gene, a dominant-acting restriction system present in many mouse strains (41,55). Fv1 encodes a Gag-like protein (4) most closely related tothe HERV-L family of endogenous retrovirus proteins andsomehow blocks the incoming viral PIC after reverse transcrip-

tion. The susceptibility of various MuLV isolates to Fv1 re-striction is largely determined by a single amino acid, residue110, of the CA protein (34). Very recently, human cells havebeen shown to exhibit a similar restriction, first dubbed Ref1activity, which blocks infection by MuLVs very early after in-fection, even before reverse transcription (29, 36). TRIM5�, amember of the so-called tripartite motif family, has been iden-tified as the protein responsible for this activity (25, 33, 53, 67,75). Remarkably, sensitivity of the MuLVs to TRIM5� is de-termined by CA and even by the same residue of the CAprotein as for Fv1 (53). These findings strongly suggest that theCA protein remains bound to the incoming viral genome andplays an important role in the early events of infection.

CA may be involved directly in nuclear entry. The simpleretroviruses typically require cell division and the associatedbreakdown of the nuclear membrane for nuclear entry (47, 57),suggesting that the PIC might find its way into the nucleus onlyby allowing the nuclear membrane to reform around it in thenewly formed daughter cells. The lentiviruses, in contrast, caninfect nondividing cells (40, 70) and thus have acquired amechanism for entry into an intact nucleus. The viral proteinsand the cellular machinery responsible for both routes of entryare uncertain. Recently, studies with chimeric viruses contain-ing different regions of the viral gag genes of Moloney MuLVand human immunodeficiency virus type 1 (HIV-1) have sug-gested that the capsid protein may be a key determinant of thedifference between these viruses (74).

Protein trafficking often involves covalent modification ofthe cargo. For example, the covalent attachment of ubiquitinor polyubiquitin chains to lysine residues of selected proteins isan important posttranslational modification that can lead to

* Corresponding author. Mailing address: Howard Hughes Med-ical Institute, Department of Biochemistry and Molecular Biophys-ics, College of Physicians and Surgeons, Columbia University, NewYork, NY 10032. Phone: (212) 305-3794. Fax: (212) 305-5106. E-mail:goff@cancercenter.columbia.edu.

† Present address: National Health Research Institutes, Division ofBiotechnology and Pharmaceutical Research, Taipai, Taiwan, ROC.

‡ Present address: Department of Genetic Medicine, Weill MedicalCollege of Cornell University, New York, NY 10021.

§ Present address: University of Texas Medical Branch, Departmentof Pathology, Galveston, TX 77555.

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the protein’s degradation or targeted transport to particularintracellular locations. A number of related molecules, includ-ing the small ubiquitin-like modifier 1 (SUMO-1; also knownas sentrin), are similarly transferred to substrate lysine residuesby conjugating enzymes (for review, see references 26, 46, and49). SUMO-1 addition involves its activation by an E1 enzymeand transfer to an E2-conjugating enzyme, Ubc9, before at-tachment to the substrate (12, 62) and is often facilitated byone of several E3 ligases (RanBP2 and PIAS1, -3, -x, and -y),which recognize the substrate and confer specificity to Ubc9(28, 31, 60). SUMO-1 is usually transferred to lysines in a Ubc9binding site motif of consensus sequence �KxE (where � is ahydrophobic residue) (48, 61), though lysines in other contextscan sometimes be modified. The functions of SUMO-1 conju-gation are not all known but can include nuclear localization(54), intranuclear movement (50), and activation of transcrip-tion factors (68). A huge number of proteins have been foundto be modified by SUMO-1 addition, including many viral tran-scriptional regulators and the Mason-Pfizer monkey virus andHIV-1 Gag proteins (19, 71). In this report, we present evi-dence that the MuLV CA protein interacts with both Ubc9 andPIASy, the E2 and E3 enzymes for SUMO-1 transfer; that CAis SUMOylated in vivo; and that these steps are required forformation of the nuclear viral DNA forms and viral replication.

MATERIALS AND METHODS

Yeast plasmids. The yeast expression vector pNLexA (Origene Technologies,Rockville, MD) and pSH2-1 (20) encode an N-terminal LexA DNA-binding domain(LexADB) and carry the HIS3 marker; the pGADNOT vector encodes a C-terminalGal4 activation domain (Gal4AD) and carries the LEU2 marker (43). The CAregion of the wild-type Mo-MuLV was synthesized by PCR on plasmid pNCS (8)with primers 5�-ACGAATTCAAAATGAAGCCCCTCCGCGCAGGAGGAA-3�and 5�-AGTCGGATCCGCAATAGCTTGCTCATCTCTCTATG-3�. The ampli-fied CA fragment was digested with EcoRI and BamHI and inserted into pNL-exA and pSH2-1 to create pMuLVCA-LexADB and pLexADB-MuLVCA con-structs, respectively. The plasmid pMuLVGAG-LexADB was prepared by fusionof the Gag coding region of Mo-MuLV to pNLexA. The Gag fragment wasamplified by PCR with primers 5�-ATCCGAATTCATGGGCCAGACTGTTACCACTC-3� and 5�-ATCCGGATCCAGTCATCTAGGGTCAGGAGGGA-3�.

The full-length Ubc9 DNA fragment was amplified by PCR with primers5�-ATCTAGGATCCAAATGTCGGGGATCGCCCTCAGC-3� and 5�-ATCTCTAGTCGACTTATGAGGGGGCAAACTTCTT-3�. The resulting PCR fragmentwas digested with BamHI and SalI and ligated with pGADNOT DNA cut withthe same enzymes to form pGal4AD-Ubc9.

The N-terminal deletion mutants of CA were created by PCR with the fixedprimer 5�-AGTCGGATCCGCAATAGCTTGCTCATCTCTCTATG-3� and oneof the following primers: dlN1 (5�-CGAATTCAAAATGCTAGTCCACTATCGCCAGTTG-3�), dlN2 (5�-CGAATTCAAAATGAATGTGTCTATGTCTTTCATT-3�), or dlN3 (5�-CGAATTCAAAATGACCCCGGAAGAAAGAGAG-3�).

The C-terminal deletion mutants of CA were created by PCR with the fixed primer5�-AACGAATTCAAAATGAAGCCCCTCCGCGCAGGAGGA-3� and one of thefollowing primers: dlC1 (5�-TCGGATCCGTTCTCGTTTATTAAAGATCTT-3�),dlC2 (5�-TCGGATCCGTTTTTTAAATCTTCTAACCT-3�), dlC3 (5�-TCGGATCCGAGTTTCTTGCCCTGGGTCCTC-3�), dlC4 (5�-TCGGATCCGGCTTCTGCCCGCGTTTTGGAG-3�), dlC5 (5�-TCGGATCCGTTGAGTGGGGCGCCCATCATC-3�), or dlC6 (5�-TCGGATCCGAACAGACTCGATCAGAGCTGT-3�).

The various resulting PCR products were digested with EcoRI and BamHIand ligated with the pNLexA vector DNA digested with the same restrictionenzymes.

Substitution mutant CA plasmids were constructed by introducing point mu-tations into the CA region of pMuLVCA-LexADB. Triple-alanine mutations werecreated by overlap extension PCR with pMuLVCA-LexADB as a template by usingoutside primers (forward primer in the N terminus of CA, 5�-AACGAATTCAAAATGAAGCCCCTCCGCGCAGGAGGA-3�; and reverse primer in the C terminusof CA, 5�-AGTCGGATCCGCAATAGCTTGCTCATCTCTCTATG-3�).

The sense-strand primers utilized for creating mutations were as follows, andthe antisense primers were their complements: CA/T1 (5�-AAGTTAGAGAGG

GCAGCTGCATTAAAAAACAAGACG-3�), CA/T2 (5�-AGGTTAGAAGATGCAGCTGCAAAGACGCTTGGAGAT-3�), CA/T3 (5�-GATTTAAAAAACGCAGCTGCAGGAGATTTGGTTAGA-3�), CA/T4 (5�-AACAAGACGCTTGCAGCTGCCGTTAGAGAGGCAG-3�), CA/T5 (5�-CTTGGAGATTTGGCAGCTGCCGCAGAAAAGATCTTT-3�), CA/T6 (5�-TTGGTTAGAGAGGCAGCTGCAATCTTTAATAAACGAGA-3�), CA/T7 (5�-GAGGCAGAAAAGGCAGCTGCAAAACGAGAAACCCCGG-3�), CA/T8 (5�-AAGATCTTTAATGCAGCTGCAACCCCGGAAGAAAGAG-3�), CA/T9 (5�-AATAAACGAGAAGCAGCTGCCGAAAGAGAGGAACGT-3�), CA/T10 (5�-GAAACCCCGGAAGCAGCTGCCGAACGTATCAGGA-3�),CA/T11 (5�-GAAGAAAGAGAGGCAGCTGCCAGGAGAGAAACAG-3�), CA/T12 (5�-CTCCAAAACGCGGCAGCTGCCCCCACCAATTTGG-3�), CA/T13 (5�-GCGGGCAGAAGCGCAGCTGCCTTGGCCAAGGTA-3�), CA/T14 (5�-AGCCCCACCAATGCAGCTGCCGTAAAAGGAATAACA-3�), CA/T15 (5�-AATTTGGCCAAGGCAGCTGCCATAACACAAGGGCCC-3�), CA/T16 (5�-GCCAAGGTAAAAGCAGCTGCCCAAGGGCCCAATGAG-3�), CA/T17 (5�-AAAGGAATAACAGCAGCTGCCAATGAGTCTCCCTCG-3�), CA/T18 (5�-CAAGGGCCCAATGCAGCTGCCCCCTCGGCCTTCCTA-3�), CA/T19 (5�-CCCAATGAGTCTGCAGCTGCCTTCCTAGAGAGACTT-3�), CA/T20 (5�-TCTCCCTCGGCCGCAGCTGCCAGACTTAAGGAAGCC-3�), CA/T21 (5�-GCCTTCCTAGAGGCAGCTGCCGAAGCCTATCGCAGG-3�), CA/T22 (5�-AAGGAAGCCTATGCAGCTGCCACTCCTTATGACCCT-3�), CA/T23 (5�-TACACTCCTTATGCAGCTGCCGACCCAGGGCAAGAA-3�), CA/T24 (5�-TATGACCCTGAGGCAGCTGCCCAAGAAACTAATGTG-3�), CA/T25 (5�-GAGGACCCAGGGGCAGCTGCCAATGTGTCTATGTCT-3�), and CA/T26 (5�-GGGCAAGAAACTGCAGCTCTATGTCTTTCATTTGG-3).

All PCR-amplified products were verified by DNA sequence analysis.The complete open reading frame for mouse PIASy was synthesized by PCR

using primers mPIASy/5�BamHI (5�-ACTACTGGATCCACATGGCGGCAGAGCTGGTGGAGGCCAAAAAC-3�) and mPIASy/3�Sal (5�-ATCTACTAGTCGACTCAGCACGCGGGCACCAGGCCTTTCTGGAA-3�). The product wascleaved with BamHI plus SalI and cloned into pGADNOT cleaved with the sameenzymes.

Yeast strains and two-hybrid library screening. Saccharomyces cerevisiaestrain CTY10-5d (MATa ade2 trp1-901 leu2-3,112 his3-200 gal4 gal80URA3::lexA-lacZ) contains an integrated GAL1-LacZ gene with LexA promoter.The yeast two-hybrid library of mouse cDNAs from WEHI-3B was describedpreviously (3). Transformation of yeast strains and scoring for LacZ expressionby 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-Gal) assay on nitrocel-lulose lifts were performed as described previously (6). Blue transformants wererestreaked and retested, and cDNA was recovered and analyzed by sequencing.

The strengths of protein-protein interactions in yeast were qualitatively as-sessed by monitoring the development of blue color after staining of colony liftsover time. The induction of reporter gene expression was measured quantita-tively using a �-galactosidase assay of permeabilized yeast grown in liquid cul-tures with ortho-nitrophenyl-�-D-galactopyranoside (ONPG) as a substrate (58).Assays were performed with cultures from three independent transformants foreach pair of constructs to be tested.

Expression of recombinant Ubc9, PIASy, and CA proteins in bacteria. Amouse Ubc9 cDNA fragment was amplified by PCR from a murine cDNApreparation using the primers 5�-ATCTAGGATCCGCCATGAGTGGGATCGCCCTCAGC-3� and 5�-ATCTAGAATTCTTATGAGGGGGCAAACTTCTT-3�.The resulting Ubc9 DNA was digested with BamHI and EcoRI and cloned invector pGEX-5X-1 (Pharmacia) to form plasmid pGEX-Ubc9. A GEX-PIASyplasmid encoding a glutathione S-transferase (GST)-PIASy fusion protein was akind gift of Rudolf Grosschedl at University of Munich. Escherichia coli strainBL21 was transformed with pGEX-Ubc9, pGEX-PIASy, and the empty vectorpGEX-6P-1 (as control) and induced by the addition of 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) for 4 h. Cells were pelleted and resuspended in10 ml of lysis buffer (10 mM Tris HCl, pH 8, 150 mM NaCl, 0.1% Triton X-100,5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 �g/ml aprotinin,1 �g/ml leupeptin, 1 �g/ml pepstatin A) containing 1 mg/ml of lysozyme. Afterincubation on ice for 1 h and brief sonication, cell debris was removed bycentrifugation at 30,000 � g for 1 h at 4°C. Five hundred microliters of gluta-thione-agarose beads (G-beads; 50% slurry) was added, and the mixture wasincubated for 1 h at 4°C and extensively washed in the same buffer. The recoveryand purity of the bound protein were examined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

For CA protein preparation, a DNA fragment containing the CA codingregion was synthesized from the wild-type viral genome of pNCA by PCR withprimers 5�-ATCCACTACCATGGCTCCCCTCCGCGCAGGAGGAAAC-3� and5�-GACTACAAGTCGACCGACAATAGCTTGCTCATCTCTCTATG-3�,digested with NcoI and SalI, and cloned into the pet21d vector (Novagene) toyield plasmid pet21d-CA. E. coli strain BL21 was transformed with pet21d-CA

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and induced and lysed by the same method as used for preparation of GST-Ubc9.One milliliter of Ni-nitrilotriacetic acid (NTA) beads (QIAGEN) was used tobind CA protein. The bound CA proteins were eluted with 100 mM imidazole.The purified CA protein was examined by 12% SDS-PAGE.

In vitro binding of CA by GST-Ubc9 and GST-PIASy beads. In a standardprotocol for binding of CA by beads containing GST fusion proteins, 20 �l of a50% (vol/vol) slurry of beads with bound GST, GST-Ubc9, or GST-PIASy (typ-ically containing 2 to 4 �g of protein) was incubated with 100 ng of purified CAprotein in 500 �l of TN buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.1% TritonX-100) containing 1 mg/ml of bovine serum albumin (Boehringer). Followingincubation for 1 h at 4°C, beads were washed three times in TN and the boundproteins were eluted by 40 mM reduced glutathione and subjected to SDS-PAGE and Western blotting.

Cell culture. 293 cells are human embryonic kidney (HEK) cells that expressthe E1 region of adenovirus 5. 293T cells are 293 cells that stably express SV40large T antigen. Rat2-2 cells are rat embryonic fibroblast cells (16). 293, 293T,and Rat2-2 cells were cultured in Dulbecco’s modified Eagle’s medium contain-ing 10% fetal calf serum.

Mammalian expression constructs. Plasmid pNCA contains an infectious copyof Mo-MuLV proviral DNA (8); pNCS is a version of pNCA that carries a simianvirus 40 (SV40) replication origin in the vector to allow high-level expression in293T cells.

Mutant plasmids were constructed by introducing point mutations into pNCS.A silent mutation was introduced to create an EcoRI site in the C terminus ofCA of pNCS by QuickChange site-directed mutagenesis kit (Stratagene) with theprimer 5�-GAAGAAAGAGAGGAACGAATTCGGAGAGAAACAGAG-3�. Theresulting construct was named pNCS/Eco.

Point mutations were created by overlap extension PCR with pNCS/Eco tem-plate DNA by using outside primers (forward primer in CA, 5�-CGCTTTTCCCCTCGAGCGCCCAGACTGGGAT-3�; reverse primer in CA, 5�-CTCCGAATTCGTTCCTCTCTTTCTTCCGG-3�). The sense-strand primers utilized for creatingmutations were as follows, and the antisense primers were their complement. Theprimers utilized for creating mutations were as follows: M1 (5�-GCATTTGCAAAACGAGAAACCCCGGAAGAA-3�), M2 (5�-ATCGCTGCAAAACGAGAAACCCCGGAAGAA-3�, M3 (5�-ATCGCTGCAGCACGAGAAACCCCGGAAGAA-3�), K193R (5�-ATTGGGAGACGATTAGAGAGGTTAGAAGATTTA-3�), K201R(5�-TTAGAAGATTTACGAAACAAGACGCTTGGAGAT-3�), K218R (5�-AAGATCTTTAATCGACGAGAAACCCCGGAAGAA-3�), K3R (5�-CCAGACATTGGGAGAAGATTAGAGAGGTTAGAAGATTTAAGAAACAGAACGCTTGGAGATTTG-3�), and K5R (5�-GACGCTTGGAGATTTGGTTAGAGAGGCAGAACGGATCTTTAATCGA-3�).

To create the pCMV2/CA construct, a CA DNA fragment was amplified byPCR with pNCS/Eco template DNA by using primers 5�-AACGAATTCAAAAATGAAGCCCCTCCGCGC-3� and 5�-AGTCGGATCCCTACAATAGCTTGCTCATCTCTCTATG-3�. The PCR products were digested by BamHI and EcoRIand inserted into the pFlag/CMV2 vector (Sigma) cut with the same restrictionenzymes. To create mutants K193R, K201R, K218R, K3R, and K5R in the contextof pCMV2/CA, two synthetic primers, 5�-AACGAATTCAAAAATGAAGCCCCTCCGCGC-3� and 5�-CTCTGTTTCTCTCCGAATTCGTTCCTCTCTTTCT-3�,were used to amplify a portion of CA by PCR with variant proviral KR mutanttemplate DNA. The amplified DNA fragment was digested by EcoRI and in-serted into the pCMV2/CA vector cut with EcoRI. All mutants created by PCRwere verified by sequencing.

Mammalian cell transfection, viral infections, and virion protein analysis. Toexamine virus viability, 293T cells were transiently transfected with wild-type(pNCS) or mutant proviral DNAs. Virus was harvested and used to infect naiveRat2-2 cells. Culture supernatants were collected on successive days and moni-tored for virus production by RT assay. To examine virion proteins, viral particleswere harvested from transfected 293T cells and purified by ultracentrifugation,and the proteins were analyzed by Western blotting as described previously (77).

In vivo SUMO conjugation assay. The plasmid for expression of the His-tagged SUMO-1 (pSG5/His-SUMO-1) was the kind gift of Anne Dejean of theInstitut Pasteur. 293T cells were grown and cotransfected with wild-type or CAmutant pCMV2/CA and pSG5/His-SUMO-1 plasmids in 140-mm-diameterdishes. Forty-eight hours posttransfection, the transfected cells were harvestedand lysed in 1 ml of lysis buffer (6 M guanidinium HCl, 100 mM NaH2PO4, and10 mM Tris-HCl, pH 7.8). After sonication, 50% of the lysate was incubated with100 �l of Ni-NTA agarose beads (QIAGEN). The beads were washed twice withwashing buffer (pH 7.8) containing 8 M urea, followed by washing with acidbuffer (pH 6.3) also containing 8 M urea. The bound proteins were eluted twicewith 8 M urea (pH 4.5) containing 20 mM imidazole. Finally, the remainingproteins were eluted with SDS-PAGE sample buffer once. The combination ofall eluates from beads was used for SDS-PAGE. A portion of the crude cell lysate

was subjected to trichloroacetic acid precipitation and used as a whole-cellextract. The proteins were analyzed by Western blotting using anti-CA antibody.

Analysis of viral DNA synthesized in vivo. Virus was prepared in 293T cells. Tominimize contamination of the virus with transfecting DNA, the cells weretransfected by Fugene 6 (Roche Applied Science, Indianapolis, IN) using only1.5 �g of DNA per dish. Forty-eight hours after transfection, the culture super-natant was harvested, assayed for RT as described previously (17, 77), andnormalized to equal RT activity. The preparations were treated with DNase I(2 �g/ml; Roche) at 37°C for 1 h to remove residual input DNA and were usedto infect naıve Rat2-2 cells. Preintegrative viral DNAs were isolated from Rat2-2cells 18 h postinfection (27) and analyzed by Southern blotting, using a 32P-labeled viral DNA probe. PCR was used to detect circular viral DNA containingtwo long terminal repeats (LTRs). The primers used to amplify the LTR-LTRjunction were MR5784 (5�-AGTCCTCCGATTGACTGAG-3�) and MR4091(5�-CTCTTTTATTGAGCTCGGG-3�) (63). The PCR conditions were 94°C for 1min, 50°C for 1 min, and 72°C for 2 min, repeated for 30 cycles. The primers usedto amplify the CA region of all viral DNAs were 5�-CCCCTCCGCGCAGGAGGAAAC-3� and 5�-CCCAGTCTGGGCGCTCGAGGGG-3�. The PCR conditionswere 94°C for 1 min, 50°C for 1 min, and 72°C for 45 s, repeated for 24 cycles.

RESULTS

The Moloney MuLV CA protein interacts with Ubc9. Bothgenetic and biochemical experiments suggest that the MuLVCA protein is involved in the trafficking or nuclear entry of theviral DNA in the early phase of the life cycle. To identifycellular proteins that might interact with CA to mediate theseprocesses, we performed a yeast two-hybrid screen. Initial testsshowed that conventional “bait” yeast plasmids expressingLexADB-CA fusions were not functional, as judged by theirinability to activate the reporter gene by homodimerizationwith a Gal4 activation domain fusion to CA. Plasmids express-ing a CA-LexADB fusion with an N-terminal CA protein anda C-terminal LexADB, however, were able to strongly dimerizewith Gal4AD-CA. This CA-LexADB “bait” was used to screenmore than 1 � 106 yeast transformants of a large library ofcDNAs from WEHI-3B, a macrophage/monocytic cell linefrom BALB/c mice (3). All of the three positive clones con-tained the complete open reading frame of Ubc9, encoding themajor SUMO-1 E2-conjugating enzyme (12, 62). The Gal4ADsequences were joined at various positions in the 5� untrans-lated portion of the Ubc9 sequences, such that translationwould proceed in each case into the Ubc9 coding region in thecorrect reading frame.

Yeast strains were transformed with various pairs of DNAsto determine the specificity of the interaction of Ubc9 and CA(Table 1). Yeast strains expressing Gal4AD-Ubc9 plus CA-LexADB showed strong activation, and those expressingGal4AD-Ubc9 plus the LexADB-CA fusion showed none, con-firming that the C-terminal CA fusion proteins were not func-tional. Gal4AD-Ubc9 also showed no interaction with a Gag-LexADB fusion protein containing the entire Gag precursor

TABLE 1. �-Galactosidase activity in yeast two-hybrid assay

Bait�-Galactosidase activity with preya:

pGal4AD pGal4AD-Ubc9 pGal4AD-mPIASy

LexADB � � �LexADB-MuLV CA � � �MuLV CA-LexADB � ��� ��MuLV GAG-LexADB � � �

a Entries depict �-galactosidase activity after colony filter lifts with X-Gal.���, colonies developing color in 1 h; ��, colonies developing color in 2 h; �,colonies remaining white after 6 h.

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protein, even though the Gag fusion protein could interactwith other partners (data not shown). These results suggestthat Ubc9 could only interact with mature CA and not with CAin the context of the immature Gag.

To test whether the interaction of Ubc9 with CA could bedetected in vitro, recombinant CA was expressed as a histidine-tagged CA protein in bacteria and purified from lysates bybinding and elution from nickel-NTA beads. Ubc9 was ex-pressed in bacteria as a GST fusion protein and was recoveredfrom lysates by binding to glutathione-agarose beads. Thebeads were then used as an affinity matrix for the binding ofCA under various conditions, and the bound proteins wereeluted and examined by SDS-PAGE followed by Western blot-ting developed with anticapsid antiserum (Fig. 1A). WhileGST control beads showed no detectable background bindingof CA, the GST-Ubc9 beads efficiently bound CA under phys-iological salt concentrations (150 mM NaCl) (Fig. 1A). Exper-iments performed in higher salt concentrations (500 mM)showed no binding, indicating that the interaction is moder-ately salt sensitive. These results suggest that the interaction isnot an artifact of the yeast system and that Ubc9 can bind CAin vitro in the absence of any other components of the trans-ferase machinery.

Identification of binding site for Ubc9 on CA. To localize theportion of the CA protein required for the interaction withUbc9, a series of deletion mutants of CA were generated,encoding truncated proteins lacking different portions of eitherthe N terminus or C terminus (Fig. 2A). The proteins wereexpressed as CA-LexADB fusions and tested for reporter ac-tivation in concert with the Gal4AD-Ubc9 fusion. CA mutantscontaining amino acids 202 to 220 showed strong activation,comparable to that of the intact CA protein, while mutantslacking this region showed little or no signal. To define thebinding sequences further, a series of substitution mutants ofCA were constructed, each encoding a protein with a block ofthree adjacent amino acids changed to alanine, spanning theessential region. The mutants were expressed as CA-LexADBfusion proteins and tested for interaction with Gal4AD-Ubc9as before. Most of the mutants still interacted strongly, butmutant T7 (with changes at amino acids 215 to 217) showedsignificantly weaker reporter activation, and mutant T8 (withchanges at amino acids 218 to 220) showed barely detectableactivation (Fig. 2B). These results suggest that residues 215 to220 (sequence IFNKRE) were important for Ubc9 binding.This sequence constitutes a near-perfect match to the knownconsensus site for Ubc9 binding and SUMOylation of its majortargets (sequence �KxE).

To extend the qualitative tests for strength of interaction,quantitative assays of the LacZ reporter gene activation for theaffected mutants were performed (Fig. 2C). The results con-firm the filter lift assays and document the strong effects of theconsensus site mutations.

CA mutations altering the Ubc9 binding site and nearbylysines block or reduce virus replication. The above muta-tional studies suggest that Ubc9 binds to CA residues 215 to220 in yeast. If this binding takes place in mammalian cells,Ubc9 would be expected to transfer SUMO-1 to one or morenearby lysine residues. Examination of the CA sequence re-vealed the presence of five lysines close to the binding site,including one within the site itself. To determine if either the

binding site or any of these lysines were important for virusreplication, a series of substitution mutations were introducedinto CA. Mutants M1, M2, and M3 contained alanine substi-tutions within the binding site, and eight more mutants con-tained changes of various of the lysines to arginines—eitherone, two, three, or all five lysines (Fig. 3A).

The various mutations were introduced into CA in the con-text of a complete infectious proviral DNA clone (pNCS), andthe mutant DNAs were then used to transform 293T cells.Culture supernatants were collected 48 h posttransfection, andthe yield of virus was estimated by assays for virion-associatedRT. All of the mutants produced levels of virus roughly similarto those of the wild type (Fig. 3B). The supernatants were thennormalized by RT levels and used to infect permissive Rat2 cells.The culture medium of these cells was collected daily, and theproduction of virus was monitored by RT assay (Fig. 3B). Whilewild-type virus was detected quickly, the binding site mutantswere all completely replication defective, with no detectable re-lease of virus at any time point. Mutants K193R and K201R andthe double mutant K(193, 201)R replicated with kinetics indis-tinguishable from those of the wild type. Mutant K218R andthe double mutants K(193, 218)R and K(201, 218)R all showeda significant delay in replication, with the first detectable virus

FIG. 1. Interaction of CA with Ubc9 and PIASy. (A) GST or GST-Ubc9 was expressed in bacteria, bound to glutathione beads, andincubated with purified recombinant CA at various salt concentrationsas indicated. Bound proteins were eluted with glutathione and ana-lyzed by SDS-PAGE and Western blotting with antisera specific forCA (�-CA) (upper panel). A duplicate gel was stained with Coomassieblue to detect the GST fusion proteins (lower panel). (B) GST orGST-PIASy was expressed in bacteria, bound to glutathione beads,and incubated with purified recombinant CA at various salt concen-trations as indicated. Bound proteins were eluted with glutathione andanalyzed by SDS-PAGE and Western blotting with antisera specific forCA (upper panel). A duplicate gel was stained with Coomassie blue todetect the GST fusion proteins (lower panel).

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appearing at day 4 rather than day 1. The identical phenotypeshown by these three mutants suggests that K218R was thesignificant mutation for all three. Finally, the K3R and K5Rmutants were almost completely replication defective, showingonly very weak signals at the latest time points. These mutants

FIG. 2. The binding of CA to Ubc9 maps to amino acids 215 to 220of CA by the yeast two-hybrid assay. (A) Deletion analysis. A sche-matic of the CA protein is shown. Numbers refer to amino acid resi-dues of CA. Yeast colonies expressing the indicated fusion proteinswere lifted and stained for �-galactosidase activity. ����, coloniesdeveloping blue color in one half-hour; ���, colonies turning blue in1 h; ��, colonies turning blue in 2 h; �, colonies turning blue in 3 h;�/�, colonies with a trace of blue color in 6 h; �, colonies with nodetectable blue color after more than 6 h. WT, wild type. (B) Alanine-scanning mutagenesis of CA. A blowup of the region of amino acids195 to 230 is shown. The position of the changes to alanine in eachmutant is as indicated. Yeast colonies expressing the indicated fusionproteins were lifted and stained for �-galactosidase activity. �-Galac-tosidase activity is indicated as in panel A. (C) Quantitative assays for�-galactosidase activity. Yeast strains expressing Gal4AD-Ubc9 andthe indicated LexA fusion proteins were grown in liquid cultures,permeabilized, and assayed for �-galactosidase activity using ONPG asa substrate. The results of three independent transformants for eachconstruct are shown.

FIG. 3. Substitution mutations in the Ubc9 binding region of CAaffect virus infectivity. (A) Amino acid sequence of the Ubc9 bindingregion of the CA protein. Numbers refer to amino acid residue. Thelysine residues are highlighted; the consensus binding site is under-lined. The position and identity of the substituted amino acids areindicated for each mutant. WT, wild type. (B, top) Production ofvirions by the mutant viruses is normal. The indicated mutant viralDNAs were introduced into 293T cells, and the release of virions intothe culture medium was assessed by measurement of virion-associatedreverse transcriptase levels. (B, bottom) Virus spreading assay. Theculture supernatants harvested from the transfected 293T cells werenormalized by RT activity and used to infect naıve Rat2-2 cells. Theculture medium from the cells infected with the various mutants wascollected on the indicated days after infection and assayed for viralspread by RT activity. (C) Western blot analysis of virion proteins ofvarious CA mutants. Virion particles were harvested from the trans-fected 293T cells and lysed, and the virion proteins were separated bySDS-PAGE, blotted, and probed with anti-CA antisera (�-CA). Thepositions of migration of the precursor Pr65Gag and the mature CAprotein are indicated.

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suggest that K218 was important for replication, but that in theabsence of K218 the other lysines contributed partial functionfor virus replication.

CA mutant viruses assemble and release normal levels ofmature virion particles. The CA mutations in the Ubc9 bind-ing site and nearby lysine residues could have effects on theexpression of the Gag proteins, Gag processing by the viralprotease, or the infectivity of the virions. To test for effects onGag expression and processing, wild-type or mutant proviralDNAs were used to transform 293T cells as before; a plasmidDNA encoding �-galactosidase was included as a control fortransfection efficiency. After 72 h, both the cells and culturesupernatants were collected. The cells were lysed, and �-galactosidase assays were used to correct for variation intransfection efficiency. The virions were purified by ultracen-trifugation through sucrose and lysed, and the virion proteinswere subjected to SDS-PAGE. The Gag proteins were visual-ized by a Western blot developed with anti-CA antisera (Fig.3C). The mutants all produced levels of virion-associated Gagproteins comparable to those of the wild-type virus and showedsimilar extents of proteolytic processing. In each case, themature p30 CA was the major species, with less Pr65Gag andtraces of a 40-kDa intermediate also present. These resultssuggest that the mutations in and around the SUMOylationconsensus motif did not significantly affect virus production.

CA lysine mutant viruses synthesize linear viral DNA butlittle or no circular DNAs. The ability of the mutant viruses toinduce the formation of normal levels of virions suggests thatthe replication-defective mutants might be blocked duringearly steps of infection. To determine the position of the block,we tested whether several of the mutants could direct thesynthesis of the various viral DNA forms. Virions were har-vested from 293T cells after transfection as before and werethen used to infect Rat2 cells. At 18 h postinfection, the low-molecular-weight DNA was harvested, separated by agarosegel electrophoresis, and blotted, and the viral DNAs weredetected by hybridization with a 32P-labeled Mo-MuLV-spe-cific probe followed by autoradiography. The wild-type virusdirected the formation of large amounts of the full-length8.8-kb linear DNA and much smaller amounts of the twocircular DNAs containing one and two copies of the viral LTR.All the mutants synthesized levels of the linear viral DNAcomparable to those of the wild type (Fig. 4A). MutantsK193R, K201R, and K218R also made normal levels of thecircular DNAs. Mutants K3R and K5R, however, made nodetectable circles. Thus, all the mutants were able to enter thecell and carry out reverse transcription normally. The replica-tion-defective viruses were blocked after reverse transcriptionand before the formation of the nuclear DNA forms. Theseresults suggest that the lysines around the SUMO-1 additionconsensus site are critical for a step after reverse transcription.

To confirm these findings, a more sensitive PCR-based assayof viral DNAs was used. The low-molecular-weight DNAsfrom the infected Rat2 cells were used as templates for PCRwith primers specific either for the LTR-LTR junction uniqueto the two-LTR circular DNA, or for the CA region present onall the viral DNAs. The reactions were initiated with twoamounts of input DNA, differing by 10-fold. The products werethen analyzed by agarose gel electrophoresis and detected byethidium stain (Fig. 4B). The wild type showed high levels of

the product of the circular DNA, mutant K218R showed lowerlevels, and mutants K3R and K5R showed no detectable cir-cular DNA. The wild-type and the mutant samples all exhib-ited similar levels of the linear DNA, with the signal responsiveto input DNA amounts. These data confirm the results of theSouthern blotting that the lysine mutants are blocked afterformation of linear DNA but before circular DNAs.

CA protein is SUMOylated in vivo. The analysis of the Ubc9binding site and lysine substitution mutants suggests that thisportion of CA plays a role early in infection and raises thepossibility that SUMO-1 modification of these CA lysinesmay be required. Direct examination of CA for SUMO-1addition in the context of the infecting virus, unfortunately,is not feasible due to the very low levels of CA present, evenafter high-multiplicity infection. To determine if CA can beSUMOylated under any circumstance, we overexpressed CAalong with a poly-histidine-tagged SUMO-1 (His-SUMO-1) in293T cells. After 24 h, lysates were prepared under stronglydenaturing conditions and proteins containing His-SUMO-1were isolated by binding to nickel-agarose beads. The boundproteins were eluted with histidine, heated in sample buffer,and separated by SDS-PAGE, and the presence of CA proteinwas detected by Western blotting with polyclonal anti-CA an-tiserum (Fig. 5). Cells expressing both CA and His-SUMO-1contained high levels of a protein detected by the anti-CA

FIG. 4. Lysine substitution mutants of CA synthesize linear viralDNA but little or no circular viral DNAs. (A) Southern blot analysis oflinear and circular viral DNA synthesis following infection of Rat2-2cells by wild-type (WT) or KR mutant viruses. Rat2-2 cells were in-fected with virus harvested from transfected 293T cells. Low-molecu-lar-weight DNA was isolated 16 h after infection, and Southern blot-ting was performed by using a radiolabeled viral DNA probe. Thepositions of linear and circular viral DNAs are indicated. (B) PCRanalysis of linear and circular viral DNA from cells infected with KRmutants. The low-molecular-weight DNA was isolated from infectedRat2-2 as in panel A and analyzed by PCR with primers specific for theLTR-LTR junction or CA region. For each mutant, either a smallamount of DNA (lanes marked 1) or a 10-times-larger amount ofDNA (lanes marked 10) was used as the input for PCR.

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antiserum at 48 kDa, the size expected for CA modified by theaddition of a single SUMO-1 moiety (lane 3). Cells expressingHis-SUMO-1 alone or CA alone did not contain this product.The formation of this species, binding to Ni-agarose and reac-tive to CA antiserum, only upon coexpression of the two pro-teins, strongly suggests CA modification. In all samples, smallamounts of free CA were detected, perhaps due to nonspecificbinding to the agarose or to cleavage of the His-SUMO-1 afterelution from the agarose.

To determine which of the lysine residues of CA were re-quired for addition of His-SUMO-1, various CA mutants wereexpressed along with His-SUMO-1, and the proteins were re-covered on Ni-agarose and analyzed by Western blotting asbefore (Fig. 5). Single-lysine mutants K193R, K201R, andK218R all showed the formation of wild-type levels of theCA-His-SUMO-1 product. Thus, no single one of these lysineswas essential. Mutant K3R, however, showed drastically re-duced levels of the addition product, and mutant K5R showedno detectable product. These results suggest that any one ofthe three to five lysines in the vicinity of the Ubc9 binding sitecould serve as an addition site, but other lysines outside thisregion could not. We cannot rule out the possibility, however,that these mutations have effects on the overall folding of CAand indirectly block the modification. Tests for the levels ofunmodified CA protein in the crude lysates showed good ex-pression from all mutants, though some (notably those con-taining K193R) contained significantly less CA protein thanthe wild type (Fig. 5, bottom). Even in these mutants, CA levelswere more than adequate for detection of the addition product(e.g., in K193R).

CA interacts with PIASy, an E3 ligase for SUMO-1 addition.While in some cases the Ubc9 E2-conjugating enzyme can me-diate SUMO-1 addition in the absence of an E3 factor, in mostcases the reaction utilizes one or another of the SUMO-spe-cific E3 ligases. We have identified one of the known E3 li-gases, PIASy (60), as interacting with another Gag-like mole-cule, the restriction factor Fv1 (manuscript in preparation). Wetherefore tested whether CA could similarly interact with PIASyin the yeast two-hybrid system. Yeasts expressing Gal4AD-PIASy and CA-LexADB did indeed show strong LacZ reporteractivation, while negative controls did not (Table 1). As forUbc9, only bait constructs containing N-terminal CA, and nei-ther C-terminal CA nor Pr65Gag, could interact with PIASy.

The interactions between the SUMO ligases and their targetproteins are often weak and transient, but specific binding cansometimes be observed in vitro. To test if the interaction ofPIASy with CA in vitro could be detected, we performed ex-periments similar to those used for Ubc9 above. RecombinantCA was prepared as before, and PIASy was expressed as a GSTfusion protein and recovered by binding to glutathione-agarosebeads. The beads were incubated with the purified CA undervarious conditions, and the bound proteins were eluted andanalyzed by SDS-PAGE followed by Western blotting (Fig.1B). The GST-PIASy beads bound CA protein under physio-logical salt concentrations, though less strongly than did GST-Ubc9 beads. The binding was not detectable in higher saltconcentrations. As before, CA did not bind to the control GSTbeads. These results suggest that PIASy does indeed bind CAin the absence of other factors in vitro.

To determine the region of CA required for the interactionwith PIASy, the series of N- and C-terminal fragments of CAwere expressed in yeast as LexADB fusion proteins along withGal4AD-PIASy, and the yeast strains were scored for reporteractivation (Fig. 6A). A single region of CA required for bind-ing was mapped to residues 132 to 173. This site is nonover-lapping with the Ubc9 site, though adjacent and immediatelyupstream from it, suggesting that Ubc9 and PIASy could bindto CA simultaneously. The PIASy binding site is almost per-fectly centered on the major homology region (MHR), themost highly conserved sequence in Gag proteins. To identifyindividual residues required for the binding, a new series ofalanine-scanning substitution mutants were generated, eachencoding a protein with a cluster of three adjacent residueschanged to alanine. These mutants were tested as CA-LexADBfusion proteins for interaction with PIASy in the yeast two-hybrid assay (Fig. 6B). Seven of the mutants (T13, T14, T16,T18, T22, T24, and T25) showed dramatically reduced bindingrelative to the wild type; one of these, T16, showed no detect-able binding. The disruptive mutations were scattered alongthe region, but included two (T16 and T18) within the MHRitself. The remaining eight mutants showed normal or evenelevated reporter activity. The results suggest that PIASy in-teracts with CA and that the binding site completely overlapswith the MHR. Importantly, all the constructs interacted nor-mally with Ubc9, indicating that the mutant proteins werestable and functional in yeast and were not grossly misfolded ordegraded in response to the mutations (Fig. 6B).

To confirm the results of the qualitative filter lift assays, thelevel of reporter gene expression was measured by quantitativeassays for �-galactosidase activity in yeast expressing various of

FIG. 5. CA is conjugated in vivo by SUMO-1. Wild-type CA and theindicated CA mutants were coexpressed in 293T cells with or withoutHis-SUMO-1 as indicated. The cells were lysed at 48 h after transfectionwith 6 M guanidine-HCl. A portion of the total extract was analyzed byWestern blotting (WB) with anticapsid antiserum (�-CA) to monitor thelevel of CA (bottom). After normalization to the amount of CA, theextracts were incubated with Ni-agarose, and the bound proteins wereeluted and analyzed by SDS-PAGE and Western blotting, developed withanticapsid antibody. Coexpression of CA and His-SUMO-1 resulted inthe appearance of a novel modified form of CA migrating at the positionexpected for the SUMO-1 conjugate (CA-His-SUMO-1). The positions ofmolecular mass markers (kDa) are indicated.

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the interaction-defective mutants (Fig. 6C). In all cases, theseassays closely supported the results of the filter lift assays.

CA mutants that fail to interact with PIASy show reducedSUMOylation in vivo. If the SUMOylation of CA by Ubc9 is anE3-dependent reaction, there should be a dependence on thebinding of the E3 enzyme to CA. To test this possibility, var-ious of the nonbinding alanine substitution mutants were ex-pressed along with His-SUMO-1 in 293T cells as before, andthe levels of modified CA-His-SUMO-1 were determined bySDS-PAGE and Western blotting (Fig. 7). Mutants T16 andT24 showed strongly reduced levels of the modified CA prod-uct, and mutant T25 showed essentially none. The total levelsof CA protein of these mutants, judged from Western blots ofthe crude lysates, were comparable to the wild type. Severalothers of the mutant CA proteins, though stable in yeast, werepoorly expressed in 293T cells and could not be tested foracceptor activity. Nevertheless, these results with the stablemutant proteins suggest that the interaction of CA with PIASyis important for efficient SUMOylation of CA by Ubc9 and thatthis is likely an E3-dependent reaction.

DISCUSSION

The experiments described above demonstrate that theMuLV CA protein interacts with the SUMO transferase ma-chinery both in yeast and in vitro and is SUMOylated in vivoand that the residues important for the interaction and modi-fication are required early in infection. The interaction of CAwith Ubc9 requires a canonical binding motif (�KxE) in theC-terminal third of the molecule. The interaction with PIASy,remarkably, seems to require a sequence directly overlappingwith the MHR, a highly conserved motif found on Gag pro-

FIG. 6. The PIASy binding site of CA lies directly on the MHR.(A) The region of CA required for binding PIASy is localized to aminoacids 132 to 173. Amino-terminal and carboxyl-terminal deletions weregenerated and expressed as LexA fusions; schematics of the retainedCA coding sequences are shown. Numbers refer to amino acid residuesof CA. Yeast colonies expressing the indicated fusion proteins werelifted and stained for �-galactosidase activity. �-Galactosidase activityis indicated as in Fig. 2A. WT, wild type. (B) Amino acid residues inthe MHR region of CA are critical for PIASy binding. The sequencefrom residue 132 to residue 173 is shown; the MHR element is under-lined. The locations of triple-alanine substitutions in the various mu-tants are indicated. Yeast colonies expressing the indicated fusionproteins were lifted and stained for �-galactosidase activity. (C) Quan-titative assays for �-galactosidase activity. Yeast colonies expressingGal4AD-PIASy and the indicated LexA fusion proteins were grown inliquid cultures, permeabilized, and assayed for �-galactosidase activityusing ONPG as a substrate. The results of three independent trans-formants for each construct are shown.

FIG. 7. Mutations that impair the binding of CA to PIASy alsoreduce SUMO-1 conjugation in vivo. Wild-type CA and the indicatedCA mutants were coexpressed with His-SUMO-1 in 293T cells. Thetransfected 293T cells were lysed with 6 M guanidine-HCl. The totalextract was analyzed by Western blotting (WB) with anticapsid anti-serum (�-CA) (bottom). After normalization to the amount of CA, theextracts were incubated with Ni-agarose, and the bound proteins wereeluted and analyzed by SDS-PAGE and a Western blot developed withanticapsid antibody. The positions of the CA-His-SUMO-1 conjugateand the molecular mass markers (kDa) are indicated.

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teins from nearly all the retroviral genera. The relative posi-tions of these two binding sites suggest that Ubc9 and PIASycould bind simultaneously to CA and cooperate to promoteSUMO transfer. There seem to be strict requirements beyondthe primary binding sites for the interaction of CA with bothproteins. Neither interacts with conventional two-hybrid pro-teins in which the N terminus of CA is blocked by fusion to theLexA DNA binding protein. This may reflect difficulties infolding of the CA protein in this context; structural studiessuggest that a free N terminus of CA might well be critical forproper folding, as the amino terminus is bound back into apocket in the mature CA proteins of several retroviruses (69).The Ubc9 and PIASy proteins also failed to interact withLexA-Gag containing the complete Gag precursor, eventhough these constructs are fully functional for the readout ofGag-Gag dimerization and interactions of Gag with other part-ners. Thus, these failures may reflect a genuine inability ofUbc9 and PIASy to access or recognize their CA binding siteswhen CA is present in the context of the precursor. The abilityof Ubc9 and PIASy to bind CA only in its mature, processed,and properly folded form would be consistent with their role inmodifying CA in the context of the PIC early in infection.

The binding of PIASy directly on the MHR is particularlyintriguing and raises the possibility that this is a critical func-tion for this highly conserved sequence element. The MHRmay have several roles in replication. Some mutants of theRous sarcoma virus and the Mason-Pfizer monkey virus withsubstitutions at the MHR are affected in virion assembly, andothers are specifically affected in early events, perhaps in thetiming of removal of CA from the incoming nucleic acid (10,66). Deletion of the entire MHR in HIV-1 causes subtle effectson Gag-Gag interactions and disrupts virus assembly, but theseeffects may be due to global changes in Gag structure (56);substitution mutations in the MHR can also affect assemblybut not Gag-Gag binding (45). For many retroviruses, the earlyfunctions of the MHR might involve its recognition by the E3ligases for SUMO-1 transfer. It is also not known whether theMHR of other simple viruses, or of the complex viruses, di-rectly interacts with any E3 ligase, nor whether SUMOylationis important for their replication. The extreme conservation ofthe MHR suggests that the process might be very widespreadamong the retroviruses. It is not clear whether PIASy is theonly E3 ligase that can recognize the Moloney MuLV CA orwhether the other known members of the family (PIAS1, -3,and -x) (7, 42, 60) could also contribute. Recently, knockoutmice lacking PIASy were generated (59, 73), and the homozy-gous mutants were found to be able to support at least basicnormal murine virus replication (73). Thus, if an E3 activity isrequired by the virus, another PIAS family member must beable to perform this role.

The residues required for the interaction of CA with Ubc9 inyeast were found to be critical for virus replication. Further-more, a number of lysine residues in the vicinity of the Ubc9consensus site were also important for virus viability. Mutationof lysine 218 to arginine in the consensus site alone signifi-cantly impaired replication, and mutation of three or five ly-sines in the region including this residue dramatically or com-pletely abolished viability. The mutations had no apparenteffect on virion assembly and release, and the resulting virusesentered cells normally and resulted in the synthesis of normal

levels of linear viral DNA. However, these mutants made sig-nificantly less or no detectable circular viral DNAs. Thus, Ubc9binding seemed to be critical for a step temporally close tonuclear entry. This step could be transport of the PIC towardthe nucleus; targeting to chromatin or nuclear componentsupon mitosis; retention of the PIC within the nucleus uponmembrane reformation; intranuclear localization of the PIC toparticular sites; or release of the DNA ends from the structureof the PIC to permit DNA circularization by host ligases.SUMOylation has been associated with nuclear targeting ofseveral proteins (54, 72), and especially with subnuclear local-ization of proteins such as the promyelocytic leukemia proteinPML to intranuclear bodies (13, 60). Perhaps SUMOylation ofCA is similarly involved in localization of the PIC to criticalsites where the viral DNA is released from the PIC, such thatboth circularization and integration can occur.

The results presented here also show that CA expressed in293T cells can indeed be modified by SUMO-1 addition invivo. The coexpression of CA and a tagged SUMO-1 resultedin readily detectable levels of a conjugate that contained thetag, was recognized with anti-CA antisera, migrated at theexpected size, and survived boiling in SDS. As seen for mostother modified proteins, the steady-state levels of the CA-SUMO-1 adduct were low, consistent with the presence ofhighly active proteases specific for removal of SUMO-1 in mostcells (21). The highest level of modification required the PIASybinding site, indicating that this is an E3-dependent reaction.Mutation of three or five lysines near the Ubc9 binding sitereduced or eliminated SUMO-1 modification, indicating that oneor more of these lysines were absolutely required in this settingand likely were serving as the addition sites. Mutation of singlelysines had no effect on the levels of the modification, suggest-ing that no one lysine served as a unique target of SUMO-1addition. Mutation K218R in particular had no effect onSUMO-1 addition, even though this single change did reducevirus replication. This discrepancy may be due to the forcedoverexpression of CA. CA is expressed at much higher levels inthese experiments than in a normal infection, and it may notrecreate the oligomeric structure of the virion core or the PIC.Thus, the sensitivity of its substrate activity for SUMOylationto mutation may not be identical to that seen in the PIC.

Taken together, these experiments suggest that the bindingand modification of CA by Ubc9 and PIASy or related E3ligases are required for the early events of infection by Molo-ney MuLV. Although Ubc9 mediates effects on some targetswithout covalent modification (35), because mutation of lysineresidues at the binding site in this case affects replication, it islikely that the actual transfer of SUMO-1 to CA is required.The step affected is close in time to nuclear entry, consistentwith the role of SUMOylation of many other substrates. Thistime is identical to that affected by mutations in another Gagprotein, p12, encoded immediately upstream of CA in the Gagprecursor (76, 77). The two Gag proteins, p12 and CA, seem toboth be required to properly ensure that the PIC enters thenucleus in a functional way early in infection, and indeed thesetwo proteins often need to be derived from the same virus tofunction properly (39). There is no indication that p12 itself ismodified by SUMO-1 or regulates CA modification.

The mechanism of nuclear entry for the MuLVs remainsobscure. These viruses, like most other simple retroviruses, do

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not efficiently infect nondividing cells, and the major block is atthe time of nuclear entry (47, 57). In dividing cells, the virusesdepend on the breakdown of the nuclear envelope associatedwith mitosis and are presumably localized inside the nuclei ofthe daughter cells as their nuclear envelopes reform. TheMuLV PICs are thus probably not imported into the nucleusthrough the activity of nuclear pore complexes, but rather throughassociation with cellular DNA, chromatin, or chromatin-associ-ated proteins. The key step may be the targeting of the PIC tonuclear components or PIC retention in the nucleus. It is alsopossible that the key step mediated by SUMOylation is afternuclear entry and involves intranuclear targeting to criticalsites or bodies within the nucleus. The addition of SUMO-1 tomany transcription factors and other target proteins for theirintranuclear localization into nuclear bodies is fully consistentwith this proposed role for SUMOylation of CA in MuLVinfection.

A number of host restriction factors that block early eventshave been found to be strongly dependent on the particularCA protein present on the incoming virus. The Fv1 gene ofmice, which provides a dominant block to infection after re-verse transcription but before appearance of the nuclear DNAforms, is a prime example (64). There are two major Fv1 alleles:Fv1n, which blocks so-called B-tropic viruses with glutamic acid atCA residue 110; and Fv1b, which blocks N-tropic viruses witharginine at this position. It is possible that the target of Fv1 actionis not only CA but actually the SUMOylation of CA. Preliminarystudies from our laboratory indicate that Fv1, like CA, inter-acts with Ubc9 and PIASy; and thus an attractive model for itsmode of action is that it might prevent the required modifica-tion of CA. The block by Fv1 is precisely at the same time inthe life cycle as the block induced by mutation of the SUMO-1modification sites, and so inhibition of CA SUMOylation byFv1 would correctly account for its time of action. In primates,a different restriction factor, the TRIM5� gene product, hasrecently been shown to block many retroviruses (25, 33, 67, 75)and again its activity depends on the CA protein of the incom-ing virus (9, 22–24, 52, 53; see references 38 and 65 for re-views). The block by TRIM5� is earlier, before reverse tran-scription, however, and so is not straightforwardly consistentwith affecting SUMOylation.

The involvement of viral proteins with the SUMO conjuga-tion system during infection is not likely to be limited to theMuLVs or even the retroviruses. The Mason-Pfizer monkeyvirus CA protein has recently been reported to interact withUbc9, and coexpression of the proteins results in their colo-calization to foci in the perinuclear region (71). Overexpres-sion of Ubc9 was found to drive a fraction of the Gag proteininto the nucleus. The p6 Gag of HIV-1 has recently beenshown to be modified by SUMO-1 addition to lysine K27,though mutation of this residue had no apparent effect on virusreplication (19). The nucleocapsid protein (NP) of the Seoul,Hantaan, and Tula hantaviruses, negative-strand RNA viruses,also interacts with Ubc9 and SUMO-1, and this interaction isrequired for localization of NP to the perinuclear region (32,37, 44). Thus, many viruses may use the SUMO-1 system forlocalization near or inside the nuclear membrane. These find-ings suggest that SUMOylation might be a prime target forintervention by antiviral inhibitors. Such new approaches may

significantly expand the range and mechanisms of action ofdrugs available for antiviral therapy.

ACKNOWLEDGMENTS

We are grateful to Valmik K. Vyas for helpful discussions and criticalreadings of early drafts. We thank Matthew Evans, Gilda Tachedjian,Scott Hughes, and Barbara Studamire for technical support and advice.

This work was supported by PHS grant R37 CA 30488 from theNational Cancer Institute. A.Y. and S.B. are Associates and S.P.G. isan Investigator of the Howard Hughes Medical Institute.

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