1 Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile
2 CNRS UMR 5097, Bordeaux, F-33000 France; Université Victor Segalen Bordeaux 2, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France
3 Bordeaux, F-33000 France; IFR 66 Pathologies Infectieuses et Cancers, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France
Correspondence
Oscar Leon
oleon{at}med.uchile.cl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the general process of integration is similar among retroviruses, there are several aspects that are not yet completely understood, especially those dealing with virus import into the nucleus. The role of viral and cellular proteins in the pathway for PIC migration to the nucleus, entry and targeting is under intense research, not only to determine possible approaches to inhibit virus replication but also to understand the mechanisms involved. For Moloney murine leukemia virus (M-MuLV), the viral proteins capsid, reverse transcriptase and IN have been reported to be PIC components (Fassati & Goff, 1999). Recently, the cellular proteins barrier-to-autointegration (BAF) and lamina-associated polypeptide 2
(LAP2
) have been described as collaborating to organize the M-MuLV PIC. For human immunodeficiency virus type 1 (HIV-1), the cellular proteins BAF (Chen & Engelman, 1998
), HMGA1 (Farnet & Bushman, 1997
) and more recently LEGF/p75 (Maertens et al., 2004
) have been shown to be important in the integration process.
Retroviral INs have essentially three functional domains [Reviewed by Chiu & Davies (2004)]. The HisHis/CysCys (HHCC) motif is highly conserved among all INs. For M-MuLV, this domain is essential for integration (Jonsson et al., 1996
) and it is also involved in protein multimerization (Yang et al., 1999
; Leon & Roth, 2000
). Some basic residues of this domain have been identified as critical for 3' processing and strand transfer (Yang et al., 2001
). In addition, the importance of the HHCC domain to the efficiency of coordinated two-end integration has been described (Yang & Roth, 2001
). The core domain or catalytic site of INs is characterized by a D-D(35)-E motif that is highly conserved among retroviruses and retrotransposons. In the case of the M-MuLV, the conserved DDE motif comprises Asp125, Asp184 and Glu220. Substitutions of any of these acidic residues in HIV-1 and Rous sarcoma virus INs result in loss of all catalytic activities (Drelich et al., 1992
; Engelman & Craigie, 1992
; Kulkosky et al., 1992
; Leavitt et al., 1993
). The C-terminal domain of INs is less conserved. This domain contributes non-specific DNA-binding activity and participates in oligomerization (Engelman et al., 1994
). IN seems to exist in a dynamic equilibrium of monomers, dimers, tetramers and high-order oligomers (Bao et al., 2003
). In vivo and in vitro complementation studies suggest that the active M-MuLV IN is a multimer (Jonsson et al., 1996
; Yang et al., 1999
; Yang & Roth, 2001
). On the basis of its amino acid sequence, the monomeric M-MuLV IN has an estimated 46 kDa molecular mass.
Most in vitro studies have been carried out with the enzyme produced in Escherichia coli. Given the high similarity between yeast and higher eukaryotes regarding protein folding, membrane trafficking and other cellular mechanisms, yeast has been widely used to uncover and establish basic aspects of fundamental biology. As a first step toward defining the cellular protein factors able to interact with IN, we used the yeast Saccharomyces cerevisiae.
In this work, we present the results of the expression and purification of M-MuLV IN in yeast. We also describe the results of the expression of M-MuLV IN in yeast strains that are deficient in RAD52 and SNF5.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Construction of the M-MuLV IN expression vector in yeast.
The yeastE. coli shuttle expression vectors used in this work were those previously described by Caumont et al. (1996). These vectors are self-replicating recombinant plasmids that contain the 2 µ replication-origin sequence, the URA3 yeast selection markers and the leu2-d genes (Erhart & Hollenberg, 1983
). These plasmids were used to transform yeast strains mutated in their LEU2 and URA3 genes. The expression vector pJV M-MuLV IN was constructed in two steps. First, the histidine-tagged M-MuLV IN expression vector pETINH1 (Jonsson et al., 1993
) was used as a template to amplify the IN coding sequence. A NcoI site at the start codon and a SalI site at the 3' terminus of the IN coding sequence were incorporated using the oligonucleotides 5'-CGCGCCATGGTAGAAAATTCATCACCCTACA-3' and 5'-CGCGGTCGACCTATAACCTTATTTTTAAGGGGTT-3', respectively. The PCR amplification was carried out using ELONGASE Enzyme Mix (Gibco-BRL). The PCR product was digested with SalI and partially digested with NcoI to obtain a 1200 bp DNA fragment.
The gel-isolated product was ligated to pBS100 digested with NcoI and SalI to directionally insert the IN gene under the control of the alcohol dehydrogenase 2/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAPDH)-inducible hybrid promoter (Cousens et al., 1987) to obtain the construct pBS100/M-MuLV IN. Positive clones were selected by the release of a 787 bp fragment by total digestion with NcoI. PBS100/M-MuLV IN was digested with BamHI and SalI to release the expected 2420 bp fragment corresponding to the ADH2/GAPDH promoter and IN, gel-isolated and ligated to a BamHI/SalI-digested pBS24.1 vector (Fig. 1
). The mutant D184A IN was constructed by overlapped PCR amplification using the mutagenic oligonucleotides 5'-TTGGGAACTGGCAATGGGCCTG-3' and 5'-CAGGCCCATTGCCAGTTCCCAA-3'. The flanking primers for amplification were the same as used for the wild-type IN. The procedure used to ligate the mutated IN to the plasmid PBS24.1 was similar to that described for wild-type IN.
|
Yeast transformation.
Yeast transformations were performed by the one-step procedure (Chen et al., 1992). Transformed cells were inoculated on YCAD solid medium lacking uracil, supplemented with 2·0 % glucose. The plates were incubated at 28 °C for 35 days.
Yeast lethality test.
The effect of M-MuLV IN expression on yeast growth on solid media was determined using a drop test (Caumont et al., 1996). Briefly, 3 µl droplets of plasmid-containing yeast standard suspensions (about 20 000 URA+ yeast cells) were dropped on YNB solid medium lacking uracil and leucine. In addition, the medium contained 0·1 % glucose, which allows high IN expression. The plates were incubated for 57 days at 28 °C and the phenotypes were observed. All lethality tests were done with IN without the His tag.
Yeast growth.
To evaluate the lethal effect of M-MuLV IN expression on yeast as a function of incubation time, liquid cultures of yeast expressing IN in a selective medium were performed. Cell growth was monitored by measuring OD600. Yeast suspensions expressing M-MuLV IN (1 ml) were prepared under the conditions described above and were inoculated in 50 ml YNB liquid medium lacking uracil and leucine. The OD600 was measured between 0 and 24 h incubation at 28 °C.
M-MuLV integrase expression and purification.
In order to analyse the expression level of M-MuLV IN, the yeast strain JSC-310 containing the expression plasmid pJV M-MuLV IN-HT was grown in 250 ml YEPD complete medium, as described by Caumont et al. (1996). After 72 h culture, yeast pellets were lysed with glass beads, as reported by Pichuantes et al. (1990)
, in a solution containing 50 mM HEPES (pH 7·4), 0·5 M NaCl, 10 mM CHAPS, 1 mM EDTA and 10 mM 2-mercaptoethanol (buffer A). Just before lysis, 1 mM PMSF, 1 mM benzamidine and 2 mg leupeptin ml1 were added. The supernatant was mixed with 0·5 ml nickelNTA agarose equilibrated with buffer A containing 15 mM imidazole. The mixture was left overnight at 4 °C in a rotator. The resin was placed in a small column and washed with 5 ml buffer A containing 20 mM imidazole. The protein was eluted with 300 mM imidazole in buffer A and 0·5 ml fractions were collected. Glycerol (0·5 ml) was added to the fractions, which were stored at 80 °C. The fractions were analysed by SDS-PAGE in 15 % polyacrylamide gels and by Western blotting using a rabbit polyclonal anti-M-MuLV IN antibody, kindly supplied by Dr M. Roth, University of Medicine and Dentistry of New Jersey. Protein was estimated by the Bio-Rad assay.
In vitro assays of M-MuLV integrase.
The 3'-processing and strand-transfer reactions contained 1 pmol labelled substrate, and the reaction buffer contained 20 mM MES (pH 6·2) 100 mM KCl, 10 mM MnCl2, 10 mM dithiothreitol and 10 % (v/v) glycerol in a final volume of 15 µl. The reaction was started by adding 15 µl IN. The reaction mixture was incubated at 37 °C for 1 h and then stopped by adding 15 µl loading buffer (95 % formamide, 20 mM EDTA, 0·05 % bromophenol blue) and heating at 90 °C for 5 min. Disintegration activity was determined in 20 mM PIPES (pH 6·4). The reaction products were analysed by electrophoresis on 12 % polyacrylamide gels with 7 M urea in Tris/borate/EDTA (TBE) buffer (pH 7·6) and autoradiographed. Substrates were labelled at the 5' end with T4 polynucleotide kinase and annealed to the complementary unlabelled strand at a ratio of 1 : 2 in 100 mM NaCl. The oligonucleotides were annealed for 3 min at 95 °C and then cooled to 25 °C. The 3'-processing and strand-transfer reactions were performed using the 5'-labelled oligodeoxynucleotides (ODNs) 5'-GATCCGACTACCCGTCAGCGGGGGTCTTTCATT-3' and 5'-GATCCGACTACCCGTCAGCGGGGGTCTTTCA-3', respectively, annealed to 5'-AATGAAAGACCCCCGCTGACGGGTAGTCGGATC-3' (Invitrogen). Disintegration assays were performed using a 5'-end radiolabelled dumb-bell substrate, as described previously (Donzella et al., 1998). The reaction products were run on 17·5 % acrylamide sequencing gels, dried, and exposed on a phosphorImager (Bio-Rad). Images were processed using Adobe Acrobat 6.0 Professional.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The protease-deficient yeast strain JSC-310 was used for expression of M-MuLV IN. The strain containing the plasmid harbouring the IN coding sequences was grown in YEPD complete medium, as indicated in Methods. The addition of a His tag at the C terminus of IN allowed its purification after a single chromatographic step. In parallel experiments we constructed a mutated IN to obtain an inactive protein. Mutation D184A was introduced into M-MuLV IN, since residue D184 has been postulated to be one of the amino acids of the putative catalytic triad of M-MuLV IN (Kulkosky et al., 1992). Both wild-type and D184A His-tagged INs were purified by chromatography on nickelNTA agarose. The purified proteins were obtained with a yield of 12 mg (l yeast culture)1.
Fig. 2(a) shows a SDS-PAGE analysis of the protein fractions. A protein band migrating near the 45 kDa marker was observed in the yeast strains that were transformed with the plasmids containing either WT IN or the mutant D184A IN (lanes 3 and 4). This protein band was not observed in a cell lysate of the JSC-310 yeast strain that was not transformed with plasmid pJV M-MuLV IN (lane 2). After elution with 300 mM imidazole, a main protein band was present for both the wild-type (lane 5) and D184A (lane 6) IN. The presence of IN in the fractions was confirmed by Western blotting (Fig. 2b
, lanes 5 and 6). In Fig. 2(b)
, lanes 2 and 3 show the proteins stained with Ponceau S (Sigma) before blotting. These results indicate that M-MuLV IN was efficiently expressed in yeast, and that the D184A mutation did not affect protein expression.
|
|
Regardless of its relevance in vivo, the disintegration reaction has been very useful in the characterization of several mutants, particularly those encoding substitutions in the core domain. We therefore assayed the disintegration activity in the presence of the dumb-bell-oligomer disintegration substrate (Fig. 3c). Only wild-type IN was able to catalyse the standard disintegration reaction (lane 2). Mutated IN showed no activity (lane 3). In contrast to human and avian retroviral INs, in which it has been experimentally shown that the DDE triad is esential for IN activity, this is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN.
Many if not all M-MuLV INs have been expressed and purified using the bacterial system. It thus seemed interesting to compare both systems, the yeast- and the E. coli-expressed INs. Fig. 4 shows a comparison of the disintegration and strand-transfer activities of the wild-type INs prepared from S. cerevisiae and E. coli. The enzyme purifed from yeast was about twofold more active than the enzyme from E. coli. This result is in agreement with the hypothesis stated above that expression of eukaryotic proteins in yeast can be more efficient.
|
The effect of M-MuLV IN expression on yeast growth was determined on selective solid medium lacking leucine under conditions that allowed high levels of IN expression (0·1 % glucose). The diploid yeast strain AB2 (d.RAD52+) and the rad52 haploid yeast strain W839-5C were transformed with the plasmid pJV M-MuLV IN. The results shown in Fig. 5(a, b), lanes 3 and 4, revealed that under these conditions expression of M-MuLV IN produced a lethal phenotype in both yeast strains. In contrast, after 5 days incubation, no lethal phenotype was observed when the yeasts were transformed with the control plasmid lacking the M-MuLV IN gene (Fig. 5
a, b, lane 1). No lethality was observed also when the yeast strains AB2 or W839-5C were transformed with the plasmid containing the inactive D184A M-MuLV IN coding sequence (Fig. 5a, b
, lane 5). Another way to detect the lethal effect produced by IN on yeast was obtained by measuring the yeast growth as a function of time. The experiment reported in Fig. 5(c)
shows that in liquid selective medium the lethal effect can also be observed when using the active M-MuLV IN.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Under defined conditions, the overexpression of M-MuLV IN produced a lethal phenotype in yeast. A similar observation has been previously described for HIV-1 IN (Caumont et al., 1996). The yeast lethal phenotype suggested that HIV-1 IN could enter into the yeast nucleus and act as a nuclease, probably cleaving chromosomal DNA (Parissi et al., 2000a
, 2003
). The mechanism of entry of HIV-1 IN into the yeast nucleus has not been completely elucidated. Maertens et al. (2004)
have reported that LEDGF/p75 is essential for nuclear localization of HIV-1 IN. Previous studies have shown that in murine cells, mitosis is required for integration of M-MuLV (Roe et al., 1993
). Since the nuclear membrane of the budding yeast is not disrupted during mitosis it is possible that M-MuLV and HIV-1 INs pass through the nuclear membrane pores to produce DNA cleavage (Barnes & Rine, 1985
).
Several lines of evidence indicate that cellular factors could affect the selection of integration sites, since host proteins could enhance or decrease integration at nearby sites (reviewed by Engelman, 2003). Yeast proteins may direct retroviral PIC to integration sites in Ty1, Ty3 (Chalker & Sandmeyer, 1992
) and Ty5 (Zou & Voytas, 1997
) retrotransposons. The requirement of the transcription factor ySNF5 (homologous to human Ini1) by both HIV-1 and M-MuLV INs to induce a lethal phenotype revealed an analogous interaction. ySNF5 is a component of the SWI/SNF chromatin-remodelling system. It is possible that ySNF5 could direct both INs to active regions of chromatin (Kalpana et al., 1994
). M-MuLV IN does not contain nuclear-localization signals and its entry into the nucleus may be determined by interaction with yeast proteins. It will be of great interest to identify those proteins.
Our results constitute the second item of evidence for lethality caused by expression of a retroviral IN in S. cerevisiae. In addition, this expression system could provide important information to search for counterparts of the natural host to define the role of cellular proteins in the import of PICs and in target-site selection. Finally, the lethal effect could also be used as a selection marker to select IN mutants, as in the case of HIV-1 (Parissi et al., 2000b).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bao, K. K., Wang, H., Miller, J. K., Erie, D. A., Skalka, A. M. & Wong, I. (2003). Functional oligomeric state of avian sarcoma virus integrase. J Biol Chem 278, 13231327.
Barnes, G. & Rine, J. (1985). Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences. Proc Natl Acad Sci U S A 82, 13541358.
Caumont, A. B., Jamieson, G. A., Pichuantes, S., Nguyen, A. T., Litvak, S. & Dupont, C. H. (1996). Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr Genet 29, 503510.[CrossRef][Medline]
Chalker, D. L. & Sandmeyer, S. B. (1992). Ty3 integrates within the region of RNA polymerase III transcription initiation. Genes Dev 6, 117128.[Abstract]
Chen, H. & Engelman, A. (1998). The barrier-to-autointegration protein is a host factor for HIV-1 integration. Proc Natl Acad Sci U S A 95, 1527015274.
Chen, D. C., Yang, B. C. & Kuo, T. T. (1992). One-step transformation of yeast in stationary phase. Curr Genet 21, 8384.[CrossRef][Medline]
Chiu, T. K. & Davies, D. R. (2004). Structure and function of HIV-1 integrase. Curr Top Med Chem 4, 965977.[CrossRef][Medline]
Chow, S. A. (1992). Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science 255, 723726.[Medline]
Cousens, L. S., Shuster, J. R., Gallegos, C., Ku, I., Stempien, M. M., Urdea, M. S., Sanchez-Pescador, R., Taylor, A. & Tekamp-Olson, P. (1987). High level of expression of proinsulin in the yeast Saccharomyces cerevisiae. Gene 61, 265275.[CrossRef][Medline]
Craigie, R. (2001). HIV-1 integrase: a brief overview from chemistry to therapeutics. J Biol Chem 276, 2321323216.
Craigie, R., Fujiwara, T. & Bushman, F. (1990). The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell 62, 829837.[CrossRef][Medline]
Donzella, G. D., Leon, O. & Roth, M. J. (1998). Implication of a central cysteine residue and the HHCC domain of Moloney murine leukemia virus integrase protein in functional multimerization. J Virol 72, 16911698.
Drelich, M., Wilhelm, R. & Mous, J. (1992). Identification of amino acids residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. Virology 188, 459468.[CrossRef][Medline]
Engelman, A. (2003). The roles of cellular factors in retroviral integration. Curr Top Microbiol Immunol 281, 209237.[Medline]
Engelman, A. & Craigie, R. (1992). Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function. J Virol 66, 63616369.[Abstract]
Engelman, A., Mizuuchi, K. & Craigie, R. (1991). HIV-1 integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell 67, 12111221.[CrossRef][Medline]
Engelman, A., Hickman, A. B. & Craigie, R. (1994). The core and carboxyl-terminal domains of the integrase protein of HIV-1 each contribute to nonspecific DNA binding. J Virol 9, 59115917.
Erhart, E. & Hollenberg, C. P. (1983). The presence of defective LEU2 gene on a 2 µ DNA recombinant plasmid of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol 156, 625635.[Medline]
Farnet, C. M. & Bushman, F. D. (1997). HIV-1 cDNA integration: requirement of HMGI(Y) protein for function of preintegration complexes in vitro. Cell 88, 483492.[CrossRef][Medline]
Fassati, A. & Goff, S. P. (1999). Characterization of intracellular reverse transcription complexes of Moloney murine leukemia virus. J Virol 73, 89198925.
Hanahan, D. (1983). Studies on transformation of E. coli with plasmids. J Mol Biol 166, 557580.[Medline]
Jonsson, C. B., Donzella, G. A. & Roth, M. J. (1993). Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli. J Biol Chem 268, 14621469.
Jonsson, C. B., Donzella, G. A., Gaucan, E., Smith, C. M. & Roth, M. J. (1996). Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis. J Virol 70, 45854597.[Abstract]
Kalpana, G. V., Marmon, S., Wang, W., Crabtree, G. R. & Goff, S. P. (1994). Binding and stimulation of HIV-1 integrase by human homolog of yeast transcription factor SNF5. Science 266, 20022006.[Medline]
Kulkosky, J., Jones, K. S., Katz, R. A., Leis, J. & Skalka, A. M. (1992). Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol 12, 23312338.[Abstract]
Leavitt, A. D., Shiue, L. & Varmus, H. E. (1993). Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. J Biol Chem 268, 21132119.
Leon, O. & Roth, M. J. (2000). Zinc fingers: DNA binding and proteinprotein interactions. Biol Res 33, 2130.[Medline]
Maertens, G., Cherepanow, P., Debyser, Z., Engelborghs, Y. & Engelman, A. (2004). Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75. J Biol Chem 279, 3342133429.
Parissi, V., Caumont, A., Richard de Soultrait, V., Dupont, C. H., Pichuantes, S. & Litvak, S. (2000a). Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast. Gene 247, 129136.[CrossRef][Medline]
Parissi, V., Caumont, A. B., de Soultrait, V. R., Calmels, C., Pichuantes, S., Litvak, S. & Dupont, C. H. (2000b). Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae. J Mol Biol 295, 755765.[CrossRef][Medline]
Parissi, V., Caumont, A., de Soultrait, V. R. & 7 other authors (2003). The lethal phenotype observed after HIV-1 integrase expression in yeast cells is related to DNA repair and recombination events. Gene 322, 157168.[CrossRef][Medline]
Pichuantes, S., Babe, L. M., Barr, P. J., DeCamp, D. L. & Craik, C. S. (1990). Recombinant HIV-2 protease processes HIV-1 pr53gag and analogous gene peptides in vitro. J Biol Chem 265, 1389013898.
Roe, T., Reynolds, T. C., Yu, G. & Brown, P. O. (1993). Integration of murine leukemia virus depends on mitosis. EMBO J 12, 20992108.[Abstract]
Suzuki, Y., Yang, H. & Craigie, R. (2004). LAP2 alpha and BAF collaborate to organize the Moloney murine leukemia virus preintegration complex. EMBO J 23, 46704678.
Turlure, F., Devroe, E., Silver, P. A. & Engelman, A. (2004). Human cell proteins and human immunodeficiency virus DNA integration. Front Biosci 9, 31873208.[Medline]
Villanueva, R. A., Jonsson, C. B., Jones, J., Georgiadis, M. M. & Roth, M. J. (2003). Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions. Virology 316, 146160.[CrossRef][Medline]
Wilhelm, M. & Wilhelm, F. X. (2001). Reverse transcription of retroviruses and LTR retrotransposons. Cell Mol Life Sci 58, 12461262.[Medline]
Yang, F. & Roth, M. J. (2001). Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase. J Virol 75, 95619570.
Yang, F., Seamon, J. A. & Roth, M. J. (2001). Mutational analysis of the N-terminus of Moloney murine leukemia virus integrase. Virology 291, 3245.[CrossRef][Medline]
Yang, F., Leon, O., Greenfield, N. J. & Roth, M. J. (1999). Functional interactions of the HHCC domain of moloney murine leukemia virus integrase revealed by nonoverlapping complementation and zinc-dependent dimerization. J Virol 73, 18091817.
Zou, S. & Voytas, D. F. (1997). Silent chromatin determines target preferences of the Saccharomyces retrotransposons Ty5. Proc Natl Acad Sci U S A 94, 74127416.
Received 1 March 2005;
accepted 7 June 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |