Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK1
Author for correspondence: Nigel Dimmock. Fax +44 2476 523568. e-mail ndimmock{at}bio.warwick.ac.uk
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Abstract |
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Introduction |
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The RT protein associated with the PIC reverse transcribes the virion RNA (for a review, see Brown, 1997 ). PICs are also responsible for transport of newly synthesized viral cDNA into the nucleus and integration of the cDNA into the target cell genome (Bukrinsky et al., 1992
; Bushman et al., 1990
). Vpr has been implicated in the targeting of PICs to the host cell nucleus but has not been shown directly to be part of the complex (Gallay et al., 1996
; Heinzinger et al., 1994
; Jenkins et al., 1998
; Karni et al., 1998
; Popov et al., 1998a
, b
; Vodicka et al., 1998
; Zhang et al., 1998
). MA is also thought to contribute to nuclear targeting of the viral cDNA (Bukrinsky et al., 1993a
; Gallay et al., 1995b
, 1996
; Heinzinger et al., 1994
; Popov et al., 1998b
; von Swedler et al., 1994
), although there is also evidence that this may not be the case (Fouchier et al., 1997
). Phosphorylation of MA may be important for its karyophilic properties (Bukrinskaya et al., 1996
; Gallay et al., 1995a
, b
). The viral IN also appears to have a role in nuclear targeting (Gallay et al., 1997
; Pluymers et al., 1999
) and possibly reverse transcription (Wu et al., 1999
), in addition to its major activity of integrating viral cDNA into the host cell genome (Bushman et al., 1990; Ellison & Brown, 1994
; Farnet & Haseltine, 1990
). Recently, it has been shown that a central DNA flap (a 99 nucleotide plus-strand overlap created during HIV-1 reverse transcription at the boundary at which left- and right-hand segments of nascent plus-strand cDNA merge), acts as a cis-determinant of HIV-1 DNA nuclear import (Zennou et al., 2000
).
Much has been done to determine the protein composition and in vitro function of PICs through the use of immunoprecipitation, integration assays, nuclear import assays and, to a certain extent, nuclease protection assays. However, less is known about the formation and maturation of PICs especially in regard to nuclear targeting, translocation and integration of viral cDNA with the host genome. Although the exact conformation of PICs is not well understood, experiments have shown that the ends of the cDNA may be joined through dimerization by the viral IN protein or the cell HMG 1(Y) protein (Ellison & Brown, 1994 ; Farnet & Bushman, 1997
; Miller et al., 1997). Recently, the proteinDNA structure of PICs partially purified from HIV-1-infected cells at 8·5 h after infection has been analysed by Mu-mediated PCR footprinting (Chen et al., 1999). The HIV-1 cDNA termini (LTRs), but not the rest of the genome, were protected from nuclease attack by bound protein. The termini form a unique structure, resembling the ends of MLV PIC DNA (Wei et al., 1997
), which suggests that this may be a common feature of retrovirus PIC DNAs.
The consensus thus far is that HIV-1 PICs contain condensed cDNA in a complex resembling a partially dissociated viral core in which proteins are tightly associated with the ends of the cDNA but only loosely associated, if at all, with the intervening sequence (Chen et al., 1999 ; Farnet & Bushman, 1997
). The work described in this report found an almost identical structure at 8·5 h post-infection using the different technique of DNase I footprinting (protease digestion followed by DNase digestion). However, PICs isolated in exactly the same way at 10 h post-infection were almost completely resistant to DNase I digestion. These late PICs were active and integrated HIV-1 cDNA into phage DNA in vitro. Thus late PICs have a complex proteinDNA structure, are functionally integrative and may represent a more mature structure than PICs present at an earlier time.
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Methods |
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Antibodies.
We used the following antibodies: mAb H12-5C to the HIV-1 CA protein (B. Chesebro and K. Wherly); rabbit antiserum to Vpr (V. Ayyavoo and D. Weiner) both obtained from the NIH AIDS Research and Reference Reagent Program, Rockville, USA; mAb 4H2B1 to MA (R. B. Ferns and R. B. Tedder); mAb IIG10E6 to RT (D. Helland and A. M. Szilvay); rabbit antiserum to IN (S. Ranjbar) all obtained from the Centralized Facility for AIDS Reagents.
Preparation of PICs.
PICs were prepared essentially as described previously (Farnet & Haseltine, 1990 ). Infection was initiated by mixing 108 syncytium-forming units of cell-free virus with 5x107 C8166 cells in 3 ml medium at 37 °C. After 5 h, 5 ml of fresh medium was added. Cells harvested at the required time were washed twice in buffer K (20 mM HEPES, pH 7·4, 5 mM MgCl2, 150 mM KCl, 1 mM dithiothreitol and 20 µg/ml aprotinin) and lysed in buffer K containing 1% Triton X-100 for 30 min at 20 °C. The Triton X-100 concentration was optimized by cell fractionation and monitored by phase-contrast microscopy. Nuclei and cell debris were removed by successive centrifugations at 1000 g for 3 min and 8000 g for 10 min. The resulting supernatant (cytoplasmic extract) was treated with 20 µg/ml RNase A for 30 min at 20 °C, and centrifuged on a 5 ml, 1530% sucrose gradient for 105 min at 149000 g (Farnet & Haseltine, 1991
). Sixteen fractions were collected from the top of the gradient. Putative PICs containing viral cDNA as judged by PCR were located in fractions 810. These co-sedimented with a 160S cowpea mosaic virus marker. To investigate viral cDNA in the infected cell nuclei, the 1000 g nuclear pellet (see above) was washed in buffer solution and Dounce homogenized in 300 µl buffer solution. Disruption of nuclei was monitored by phase contrast microscopy. Lysed nuclear extracts were proteinase K treated (1 mg/ml; Roche Molecular Biochemicals, PCR grade) for 1 h at 55 °C. DNA was phenolchloroform extracted and recovered by precipitation with ethanol overnight at -20 °C. PCR involved preheating in a Touchdown PCR instrument (Hybaid) at 94 °C for 3 min, denaturing at 94 °C for 45 s, annealing at 56 °C for 45 s and an elongation step at 72 °C for 1 min. A final elongation step at 94 °C for 10 min was performed. DNA was subjected to PCR for 30 cycles. PCR products were analysed by electrophoresis on 3% agarose gels. Experiments were carried out in duplicate on different lots of infected cells.
Immunoprecipitation of PICs from sucrose velocity gradient fractions.
Fifty µl of sucrose gradient fractions containing detectable 160S cDNA were incubated overnight at 4 °C with 10 µl of monoclonal antibodies against RT, MA, CA at 10 µg/ml or 10 µl of polyclonal antibodies (1/300) against IN or Vpr. Next antibodyantigen complexes were collected for 2 h at room temperature using either protein A or G Sepharose beads (SigmaAldrich) in their respective binding buffer (50 mM Tris, 150 mM NaCl, pH 8·0 or 0·01 M NaH2PO4, 0·15 M NaCl, 0·01 M EDTA, pH 7·0). Beads with bound complexes were then washed three times in wash buffer (10 mM TrisHCl, pH 7·4, 150 mM NaCl and 1% Triton X-100). These were then digested with proteinase K at 1 mg/ml for 1 h at 55 °C to release immunoprecipitated complexes from the Sepharose beads and the beads were removed by centrifugation. The supernatant containing any viral cDNA was phenolchloroform extracted, ethanol precipitated and amplified by PCR as described above using the GAG2 primer pair (see below and Table 1) and 30 cycles of amplification.
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DNase I footprinting.
PICs were treated with DNase I (RNase free; SigmaAldrich) at 0·1, 1, 10 and 30 µg/ml for 30 min at 37 °C, and then reacted with proteinase K (1 mg/ml) at 55 °C for 1 h to remove protein. DNA was phenolchloroform extracted and precipitated with ethanol overnight at -20 °C. PCR was then carried out for 24, 26, 28 and 30 cycles using 23 primer pairs designed by Primer Designer for Windows (Version 3.0; Scientific and Educational Software) from the sequence of HxB2. These covered the entire HIV-1 genome in approximately 500 bp overlapping fragments (Table 1). PCR conditions were optimized for each primer pair so that each had specific MgCl2 and primer concentrations in the PCR mix. Positive controls were amplified following proteinase K treatment (1 mg/ml) but in the absence of DNase I and negative controls (-) were amplified following proteinase K and then DNase I treatment at 0·1 µg/ml. Footprinting was carried out twice on PIC preparations from different batches of infected cells.
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Results |
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PICs were harvested from C8166 cells infected with cell-free virus and harvested at 8·5 h post-infection. Cytoplasms were prepared and fractionated by sucrose velocity gradient centrifugation. PICs sedimenting at 160S were subjected to DNase footprinting using four concentrations of DNase I (0·1, 1, 10 and 30 µg/ml). Digestion with proteinase K was then carried out and protected DNA amplified for 24, 26, 28 and 30 cycles with 23 primer pairs that covered the entire HIV-1 genome. After analysis on agarose gels it was clear that PCR products were only seen with primer pair LTR1 and with 10, 1 and 0·1 µg/ml DNase I (Fig. 3). No PCR product was seen using any of the other 22 primer pairs, even with 30 cycles of PCR, or at any DNase concentration. The negative result shown with GAG4 primers was representative of data with the other 21 primer pairs (Fig. 3
). PCR was continued up to 36 cycles in combination with the lowest DNase I concentration, but still no product was seen (data not shown). Positive controls (+) were successfully amplified after protease treatment (1 mg/ml) but in the absence of DNase I (Fig. 3
), and no amplicon was seen in negative controls (-) following proteinase K and DNase I treatment (0·1 µg/ml). These data showing protection of only part of the LTR(s) are entirely consistent with Mu-mediated PCR footprinting on PICs harvested at a similar time (Chen et al., 1999
).
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Discussion |
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The main thrust of this report is the investigation of DNAprotein interactions over the entire HIV-1 genome using DNase I footprinting combined with PCR. In total, 23 overlapping sequences from the 5' LTR to the 3' LTR were probed and we examined PICs isolated at 8·5 and 10 h after infection of C8166 cells with cell-free virus. In the 8·5 h PICs, we found that only part of the LTR (region 1, amplified with primer pair LTR1) was protected from DNase I (Fig. 3). This amounted to approximately 6% of the cDNA, and is entirely consistent with earlier data (Chen et al., 1999
). Together these data suggest that very limited amounts of protein are tightly bound to the cDNA of 8·5 h PICs. However, when we analysed PICs isolated at 10 h post-infection in exactly the same way, a completely different picture emerged. Here nearly all (approximately 90%) of the viral cDNA genome was protected by bound protein from digestion by DNase I. The fact that the one protected region 1 (LTR1) in 8·5 h PICs (Fig. 3
) was amplified to a similar extent in the 10 h sample (Fig. 5
) underlines the significance of areas of higher nuclease resistance seen elsewhere in the genome. However, not all the 10 h PIC cDNA genome was protected from DNase digestion and region 20 was completely sensitive (Fig. 5
). Regions of high protection did not correspond with either of the two genomic polypurine tracts (Charneau et al., 1994
).
As HIV-1 infection of cells in culture comprises a series of multiple non-synchronous infection events, we cannot fully discount the fact that the 10 h PICs may comprise mixtures of structures at various states of maturity that have been derived from virions entering the cell asynchronously. However, data in Fig. 1(a, b
) suggest that there is synchronous movement of PIC cDNA from cytoplasm to nucleus. In addition, the 8·5 and 10 h post-infection patterns of nuclease protection differ radically as shown above, and in two separate experiments the nuclease protection findings were reproducible even to the extent of nuclease concentration sensitivity and number of PCR cycles required. Such data suggest that the characteristics of infection did not vary significantly from infection to infection. The positive controls shown in Fig. 5
suggest that the PICs isolated at 10 h post-infection represent the PIC population as a whole, and thus they may be an intermediate on the pathway into the nucleus. Further, PICs described here are unlikely to be proviral contaminants, as in our system, as discussed above, there was no detectable cross-contamination of cytoplasm and nucleus. In addition, our 10 h PICs were well characterized as originating from a cytoplasmic fraction that sedimented at 160S (Bowerman et al., 1989
; Farnet & Haseltine, 1990
; Karageorgos et al., 1993
; Miller et al., 1997
) and being associated with the viral proteins RT, IN, Vpr and MA, but not CA, as others have also found (see Introduction for references). It is interesting that there was no difference in the types of virion protein detected in our 8·5 and 10 h PICs. Clearly, the increased nuclease resistance at 10 h could only be explained if the distribution or association of PIC-associated proteins had changed as hypothesized in Fig. 6(a
c
), or more (possibly cell) proteins had been recruited (Fig. 6d
).
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In conclusion, we have described a new form of nuclease-resistant PIC present at 10 h post-infection that appears late in the cytoplasm. This may be a mature and integration-competent intermediate that appears just prior to nuclear membrane docking and translocation. More work is needed to support this view, and we are currently engaged in identifying proteins that are bound near the central DNA flap of the late PIC, and investigating if and how they might aid nuclear translocation.
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Acknowledgments |
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References |
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Bowerman, B., Bishop, P. O., Bishop, J. O. & Varmus, H. E. (1989). A nucleoprotein complex mediates the integration of retroviral DNA. Genes & Development 3, 469-478.[Abstract]
Brown, P. O. (1997). Integration. In Retroviruses, pp. 161203. Edited by J. Coffin, S. H. Hughes & H. E. Varmus. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Bukrinskaya, A. G., Ghorpade, A., Heinzinger, N. K., Smithgall, T. E., Lewis, R. E. & Stevenson, M. (1996). Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA. Proceedings of the National Academy of Sciences, USA 93, 367-371.
Bukrinsky, M. I., Sharova, N., Dempsey, M. P., Stanwick, T. L., Bukrinskaya, A. G., Haggerty, S. & Stevenson, M. (1992). Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proceedings of the National Academy of Sciences, USA 89, 6580-6584.[Abstract]
Bukrinsky, M. I., Haggerty, S., Dempsey, M. P., Sharova, N., Adzhubel, A., Spitz, L., Lewis, P., Goldfarb, D., Emerman, M. & Stevenson, M. (1993a). A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365, 666-669.[Medline]
Bukrinsky, M. I., Sharova, N., McDonald, T. L., Pushkarskaya, T., Tarpley, W. G. & Stevenson, M. (1993b). Association of integrase, matrix and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proceedings of the National Academy of Sciences, USA 90, 6125-6129.[Abstract]
Bushman, F. D., Fujiwara, T. & Craigie, R. (1990). Retroviral integration directed by HIV integration protein in vitro. Science 249, 1555-1558.[Medline]
Charneau, P., Mirambeau, G., Roux, P., Paulous, S., Buc, H. & Clavel, F. (1994). HIV-1 reverse transcription: a termination step at the centre of the genome. Journal of Molecular Biology 241, 651-662.[Medline]
Chen, H., Wei, S.-Q. & Engelman, A. (1999). Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type 1 intasome. Journal of Biological Chemistry 274, 17358-17364.
Ellison, V. & Brown, P. O. (1994). A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro. Proceedings of the National Academy of Sciences, USA 91, 7316-7320.[Abstract]
Farnet, C. M. & Haseltine, W. A. (1990). Integration of human immunodeficiency virus type 1 DNA in vitro. Proceedings of the National Academy of Sciences, USA 87, 4164-4168.[Abstract]
Farnet, C. M. & Haseltine, W. A. (1991). Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. Proceedings of the National Academy of Sciences, USA 65, 1910-1915.
Farnet, C. M. & Bushman, F. D. (1997). HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro. Cell 88, 483-492.[Medline]
Fouchier, R. A. M., Meyer, B. E., Simon, J. H. M., Fischer, U. & Malim, M. H. (1997). HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import. EMBO Journal 16, 4531-4539.
Fujiwara, T. & Mizuuchi, K. (1988). Retroviral DNA integration: structure of an integration intermediate. Cell 54, 497-504.[Medline]
Gallay, P., Swingler, S., Aiken, C. & Trono, D. (1995a). HIV-1 infection of non-dividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell 80, 379-388.[Medline]
Gallay, P., Swingler, S., Song, J., Bushman, F. & Trono, D. (1995b). HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell 83, 569-576.[Medline]
Gallay, P., Stitt, V., Munday, M., Oettinger, M. & Trono, D. (1996). Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import. Journal of Virology 70, 1027-1032.[Abstract]
Gallay, P., Hope, T., Chin, D. & Trono, D. (1997). HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karopherin pathway. Proceedings of the National Academy of Sciences, USA 94, 9825-9830.
Heinzinger, N. K., Bukrinsky, M. I., Haggerty, S., Ragland, A. M., Kewelramani, V., Lee, M.-A., Gendelman, H. E., Ratner, L., Stevenson, M. & Emerman, M. (1994). The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proceedings of the National Academy of Sciences, USA 91, 7311-7315.[Abstract]
Jenkins, Y., McEntee, M., Weis, K. & Greene, W. C. (1998). Characterization of HIV-1 Vpr nuclear import: analysis of signals and pathways. Journal of Cell Biology 143, 875-885.
Karageorgos, L., Li, P. & Burrell, C. J. (1993). Characterization of HIV replication complexes early after cell-to-cell infection. AIDS Research and Human Retroviruses 9, 817-823.[Medline]
Karni, O., Friedler, A., Zakai, N. & Loyter, A. (1998). A peptide derived from the N-terminal end of HIV-1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr. FEBS Letters 429, 421-425.[Medline]
Levy, J. A. (1998). HIV and the Pathogenesis of AIDS, 2nd edn. Herndon, VA: ASM Press.
Li, P. & Burrell, C. J. (1992). Synthesis of human immunodeficiency virus DNA in a cell-to-cell transmission model. AIDS Research and Human Retroviruses 8, 253-259.[Medline]
McLain, L. & Dimmock, N. J. (1994). Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies. Journal of General Virology 75, 1457-1460.[Abstract]
Miller, M. D., Farnet, C. M. & Bushman, F. D. (1997). Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. Journal of Virology 71, 5382-5390.[Abstract]
Pluymers, W., Cherepanov, P., Schols, D., de Clercq, E. & Debyser, Z. (1999). Nuclear localization of human immunodeficiency virus type 1 integrase expressed as a fusion protein with green fluorescence protein. Virology 258, 327-332.[Medline]
Popov, S., Rexach, M., Ratner, L., Blobel, G. & Bukrinsky, M. (1998a). Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex. Journal of Biological Chemistry 273, 13347-13352.
Popov, S., Rexach, M., Zybarth, G., Reiling, N., Lee, N.-A., Ratner, L., Lane, C. M., Moore, M. S., Blobel, G. & Bukrinsky, M. (1998b). Virus protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO Journal 17, 909-917.
Popovic, M., Sarngadharan, M. G., Read, E. & Gallo, R. C. (1984). Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-500.[Medline]
Pryciak, P. M. & Varmus, H. E. (1992). Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection. Cell 69, 769-780.[Medline]
Salahuddin, S. Z., Markham, P. D., Wong-Staal, F., Franchini, G., Kalyanaraman, V. S. & Gallo, R. C. (1983). Restricted expression of human T-cell leukemia-lymphoma virus (HTLV) in transformed human umbilical cord blood lymphocytes. Virology 129, 51-64.[Medline]
Vodicka, M. A., Koepp, D. M., Silver, P. A. & Emerman, M. (1998). HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes & Development 12, 175-185.
von Swedler, U., Jornbluth, R. S. & Trono, D. (1994). The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proceedings of the National Academy of Sciences, USA 91, 6992-6996.[Abstract]
Wei, S.-Q., Mizuuchi, K. & Craigie, R. (1997). A large nucleoprotein complex assembly at the ends of the viral DNA mediates retroviral integration. EMBO Journal 16, 7511-7520.
Whitcomb, J. M. & Hughes, S. H. (1992). Retroviral reverse transcription and integration: progress and problems. Annual Review of Cell Biology 8, 275-306.
Whitwam, T. & Poeschla, E. M. (2001). Identification of central DNA flap in feline immunodeficiency virus. Journal of Virology 75, 9407-9414.
Wu, X., Liu, H., Xiao, H., Conway, J. A., Hehl, E., Kalpana, G. V., Prasad, V. & Kappes, J. C. (1999). Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex. Journal of Virology 73, 2126-2135.
Zennou, V., Petit, C., Guetard, D., Nehbass, U., Montagnier, L. & Charneau, P. (2000). Genome nuclear import is mediated by a central DNA flap. Cell 101, 173-185.[Medline]
Zhang, S., Pointer, D., Singer, G., Feng, Y., Park, K. & Zhao, L.-J. (1998). Direct binding to nucleic acids by Vpr of human immunodeficiency virus type 1. Gene 212, 157-166.[Medline]
Received 18 March 2002;
accepted 25 June 2002.