Department of Molecular and Structural Biology, Aarhus University, , Denmark1
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands2
Author for correspondence: Ben Berkhout. Fax +31 20 6916531. e-mail b.berkhout{at}amc.uva.nl
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Abstract |
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Introduction |
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Retroviral RNA dimerization affects the virus life cycle at several stages. Mutation of the DIS region results in reduced packaging of viral RNA (Berkhout & van Wamel, 1996 ; Haddrick et al., 1996; Laughrea et al., 1997
; Sakuragi & Panganiban, 1997
). Surprisingly, RNA isolated from these mutant virions is predominantly dimeric, indicating that there may be additional dimerization signals. These results may also indicate that RNA dimers are packaged preferentially over monomeric RNA and it has been proposed that the DIS functions directly as a packaging signal (Clever & Parslow, 1997
; McBride & Panganiban, 1996
). Dimeric RNA allows for template switching during the process of reverse transcription, e.g. when the viral RNA template is damaged or when its secondary structure induces stalling of the reverse transcriptase enzyme (reviewed by Gotte et al., 1999
; Negroni & Buc, 2001
). Such recombinations promote genetic diversity and, in the event that different viral genomes are co-packaged, the formation of recombinant retroviruses with novel properties.
Mosaic retroviruses that result from recombination between different subtypes of HIV-1 have been reported and the same is true for HIV-2 subtypes (Bobkov et al., 1998 ; Carr et al., 1996
, 1998
; Gao et al., 1994
; Piyasirisilp et al., 2000
). Recombination can also take place between HIV-1 isolates from the distinct groups M and O, which have a limited sequence similarity of 65% (Peeters et al., 1999
; Takehisa et al., 1999
). Thus far, no recombinants between HIV-1 and HIV-2 have been reported. The creation of new recombinants is likely to depend, among other things, on the possibility of forming and packaging heterodimeric RNA genomes. In this study, we have examined the requirements for in vitro heterodimerization of the DIS-containing leader transcripts of HIV-1 and HIV-2. We report that an identical palindrome sequence, similar DIS loop size and/or palindrome orientation are essential requirements for KL heterodimerization, but formation of ED dimers is completely restricted.
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Methods |
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In vitro dimerization assays.
In the Mg2+ titration experiment, we used approximately 2 pmol of radiolabelled transcripts. The RNA was incubated for 10 min at 65 °C in dimerization buffer with 10 mM TrisHCl pH 7·5, 40 mM NaCl and either 0·1, 1 or 5 mM MgCl2 and slowly cooled to room temperature. Each sample (10 µl) was mixed with 5 µl native gel loading buffer (30% glycerol with bromophenol blue dye) and analysed on 4% native polyacrylamide gels containing either 0·25x TBE or 0·25x Trisborate0·1 mM MgCl2 (TBM). Gels were run at 150 V at room temperature, followed by drying and autoradiography. In the other experiments, approximately 2 pmol of radiolabelled RNA was first incubated, either separately or mixed, for 2 min at 85 °C in H2O and immediately placed on ice. Dimerization buffer (10x) was added to make a final reaction mixture of 10 µl with 5 mM MgCl2, 10 mM TrisHCl pH 7·5 and 40 mM NaCl. For investigation of KL dimer formation, samples were incubated for 30 min at 37 °C. ED dimers were formed by incubation for 10 min at 65 °C and slow cooling to room temperature. Electrophoresis was performed in 0·25x TBE and TBM gels followed by drying and autoradiography.
MFOLD RNA secondary structure predictions.
The sequences of the HIV-1 LAI and HIV-2 ROD isolates were downloaded from the HIV database (http://hiv-web.lanl.gov/). Secondary structure prediction was performed with the MFOLD algorithm, version 3.0 (Mathews et al., 1999 ), on the MBCMR MFOLD server (http://mfold.edu.burnet.au/) and analysed with standard settings. The thermodynamic stability values (
G, kcal/mol) presented in Table 2
are based on MFOLD analysis with model hairpin templates, of which the stem is composed of the two interacting sequences in the KL and ED dimers. We used the following model substrates: HIV-1 DIS as in Figs 1
and 4
, with seven consecutive base pairs, and HIV-2 DIS as in Fig. 4
(not the extended format as shown in Fig. 1
). It is obvious that this approach only provides an estimate of the thermodynamic stability of the KL and ED dimers. This method scores only the predicted WatsonCrick base pair interactions but will miss all non-canonical interactions that are likely to exist in both the KL and the ED dimer complexes.
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Results |
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The MgCl2 requirement for dimerization of the HIV-1 and the GC3 mutant of HIV-2 (HIV-2GC3) transcripts was investigated. Approximately 2 pmol of each radiolabelled transcript was incubated in dimerization buffer with either 0·1, 1 or 5 mM MgCl2 for 10 min at 65 °C, followed by slow cooling of the samples to room temperature and analysis on a native TBM gel (Fig. 2). Dimerization of the HIV-2GC3 transcript is barely detectable at 0·1 mM MgCl2 (Fig. 2
, lane 3) and approximately 50% dimerization is observed at 5 mM MgCl2 (Fig. 2
, lane 13). The HIV-1 transcript remains dimerization-inactive at all Mg2+ concentrations tested (Fig. 2
, lanes 1, 6 and 11): this transcript is dimerization-impaired because it folds into an RNA conformation that masks the DIS motif (Huthoff & Berkhout, 2001
). Therefore, we also synthesized the A mutant transcript (Das et al., 1997
), which contains a stabilized poly(A) hairpin that favours folding of a dimerization-competent leader RNA conformation that exposes the DIS hairpin (Fig. 1
). This A mutant displays efficient dimerization at 0·1 mM MgCl2 (Fig. 1
, lane 2) and 100% dimer was obtained at 1 and 5 mM MgCl2 (Fig. 1
, lanes 7 and 12).
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In a search for conditions that would allow RNA heterodimerization, we incubated the transcripts at conditions that promote the formation of the KL or ED dimer (Laughrea & Jette, 1996 ; Muriaux et al., 1996b
). We have used the HIV-1 A mutant and HIV-2GC3 transcripts, which homodimerize efficiently in all subsequent experiments; these transcripts are denoted HIV-1 and HIV-2 for simplicity. The HIV-1 and HIV-2 transcripts were incubated individually or in combination. To disrupt any homodimers that might have formed during the initial RNA renaturation step of purified T7 transcripts, we first mixed the two RNAs and denatured them by incubation in water for 2 min at 85 °C, followed by immediate chilling on ice. Transcripts were then incubated in dimerization buffer (5 mM MgCl2 and 40 mM NaCl), either for 30 min at 37 °C to promote KL dimer formation or for 10 min at 65 °C and slow cooling to room temperature to promote ED dimer formation. Samples were subjected to electrophoresis on TBM and TBE gels (Fig. 3A
, B
, results are summarized in Table 2
). The TBM gel allows the detection of KL dimers because the presence of Mg2+ ions prevents disruption of these relatively instable dimers during electrophoresis. The efficiency of KL homodimerization is approximately 80 and 20% for HIV-1 and HIV-2 RNA, respectively (Fig. 3A
, lanes 1 and 2), whereas both transcripts form ED homodimers with an efficiency of approximately 20% (Fig. 3B
, lanes 4 and 5). Most importantly, it is apparent that neither KL nor ED heterodimer formation occurs under the assay conditions used. These results indicate that the sequence or structural differences between the HIV-1 and the HIV-2 DIS elements are too severe to allow heterodimerization, even though we have inserted the homologous GC3 palindrome in HIV-2.
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This set of HIV-2 DIS mutants was tested in dimerization assays with or without HIV-1 RNA (Fig. 5, results are summarized in Table 2
). After heat denaturation, approximately 2 pmol of the HIV-1 transcript and the mutant HIV-2 transcripts were incubated individually (Fig. 5
, lanes 17) or in HIV-1/HIV-2 combinations (Fig. 5
, lanes 813). Samples were incubated for 30 min at 37 °C in dimerization buffer (5 mM MgCl2 and 40 mM NaCl) and subsequently analysed by electrophoresis on a TBM gel to detect KL-type dimers. As expected, all transcripts can form KL homodimers (Fig. 5
, lanes 17). No KL heterodimers were formed between HIV-1 RNA and the HIV-2 mutants +A and -A (Fig. 5
, lanes 9 and 10) but a faint heterodimer band is apparent in combination with the HIV-2 mutants -2A, -2AM and -2AMM (Fig. 5
, lanes 1113, marked D12). This result indicates that an identical DIS loop size and/or palindrome orientation is required for KL heterodimer formation. We also analysed these samples with the ED dimer protocol on a TBE gel and, whereas ED homodimer formation occurs with similar efficiencies for all HIV-2 mutants, we did not observe any ED heterodimers (results not shown).
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We measured no ED heterodimer formation between HIV-1 RNA and any of the HIV-2 mutants (results not shown). It is possible that the remaining sequence differences in the DIS hairpin impose restrictions on the formation of ED heterodimers. This idea is substantiated by MFOLD modelling of the thermodynamic stability of the predicted RNA complexes. A thermodynamic stability of approximately -30·0 kcal/mol is predicted for ED heterodimers between HIV-1 RNA and the set of HIV-2 transcripts containing the GC3 palindrome, except for the -2AM and -2AMM mutants (Table 2). However, the homodimers can form much more stable ED complexes (-40·3 and -34·7 kcal/mol for HIV-1 and HIV-2, respectively). Conversion of KL dimers into ED dimers requires melting of the stem region of two DIS hairpins, corresponding to a thermodynamic stability of -13·3 and -11·9 kcal/mol per hairpin for HIV-1 and HIV-2, respectively. For the ED homodimers, there will be a significant gain of thermodynamic stability, which will shift the KLED equilibrium to the right. The ED heterodimers are predicted to achieve a more intermediate thermodynamic stability of approximately -30·0 kcal/mol, which apparently is not sufficient to drive the KL to ED conversion.
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Discussion |
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This in vitro study indicates that heterodimerization of wild-type HIV-1 and HIV-2 RNA genomes does not readily occur. We have observed that DIS-mediated homodimerization is highly favoured over heterodimerization. However, these results do not preclude that heterodimerization may take place by a DIS-independent mechanism. For instance, dimerization of the HIV-1 genome has been reported to occur in vivo in the absence of a functional DIS element, suggesting that additional dimerization signals exist within retroviral genomes (Berkhout & van Wamel, 1996 ; Haddrick et al., 1996
; Laughrea et al., 1997
). Consistent with this idea, it was described that the presence of non-homologous DIS elements from different HIV-1 subtypes is not a principal obstacle to intersubtype recombination (St Louis et al., 1998
). If the additional dimerization signals are compatible between the genomes of HIV-1 and HIV-2, the occurrence of heterodimerization in vivo may not be as rare as predicted by our in vitro experiments.
Despite extensive recombination within the HIV-1 group and within the HIV-2 group, no HIV-1/HIV-2 recombinants have been described thus far: this is not simply due to the lack of HIV-1/HIV-2 co-infections, for which there are multiple reports (Evans et al., 1988 ; George et al., 1992
; Peeters et al., 1992
). The non-appearance of HIV-1/HIV-2 recombinants may be related to the apparent inability to make RNA heterodimers but incompatibility problems may also arise at other levels. Packaging of the heterodimeric RNA genome into virions may present a bottleneck. Primate lentiviruses can cross-package each other's RNA genome, e.g. HIV-1 is capable of packaging HIV-2 RNA (Kaye & Lever, 1998
), although the reverse is not possible, and there is reciprocal cross-packaging between HIV-1 and simian immunodeficiency virus (Rizvi & Panganiban, 1993
; White et al., 1999
). Although efficient cross-packaging of genomic RNA occurs between different lentiviruses, this is not necessarily identical to the packaging of a heterodimeric RNA genome, which is the key event that must take place prior to recombination. Because dimerization of retroviral RNA may be closely linked to the encapsidation process (Berkhout & van Wamel, 1996
; Clever & Parslow, 1997
; Lever, 2000
), it is quite possible that the inability to form heterodimers between HIV-1 and HIV-2 RNA will also inhibit co-packaging. Even if a heterodimeric RNA genome is packaged successfully, there may be additional hurdles on the way to a recombinant virus. For instance, reverse transcription may be aborted because it requires identical repeat sequences in the long terminal repeat elements. Furthermore, the recombination rate may be affected directly for co-packaged retroviral RNA dimers with a suboptimal DIS interaction (Balakrishnan et al., 2001
; Lund et al., 1999
). The likelihood of creating a viable recombinant virus is further restricted by the complexity of the lentiviral genomes. The lentiviral genome encodes certain combinations of genetic elements that may loose their function when detached by recombination. For instance, the TatTAR and RevRRE interactions (Berkhout et al., 1990
; Dillon et al., 1990
; Emerman et al., 1987
; Malim et al., 1989
) may hinder the generation of viable HIV-1/HIV-2 recombinants.
It has been suggested that at least 10% of the HIV variants currently isolated may have mosaic genomes due to recombination (Robertson et al., 1995 , 2000
). Sequential recombination events, in which an early recombinant serves as a parental strain for subsequent recombinations, have also been described (Salminen et al., 1997
). Two distinct DIS palindromes are present among the different HIV-1 subtypes of the major group M: GCGCGC is present in subtypes B and D and GUGCAC in subtypes A, C, E, G, H and J. Both DIS palindromes have been described for subtype F viruses but GCGCGC is found more frequently. Interestingly, we identified both palindrome sequences in an individual infected with a subtype F virus (E. Andersen, R. Jeeninga, B. Berkhout and J. Kjems, unpublished results). The GUGCAC palindrome is also present in viruses of the outlier group O and the new group N. Because the sequence of the palindrome may influence RNA heterodimerization, we investigated whether the known recombination events occurred preferentially between HIV-1 subtypes with the same DIS palindrome. Inspection of the HIV sequence database (http:/hiv-web.lanl.gov/CRFs/CRFs.html) indicates that most of the circulating recombinants have putative parents with an identical DIS palindrome. It will be of interest to perform a more in-depth study once more recombinant viruses have been sequenced. We realize that these recombination patterns may have been strongly influenced by other factors, most notably the requirement for the two parental viruses to share a habitat.
The results presented in this study are also relevant for safety issues concerning the use of retrovirus vectors for gene therapy. Besides HIV-1, other lentiviruses, including HIV-2, have been proposed as gene therapy vectors (Lever, 2000 ; Naldini et al., 1996
). This raises the possibility of interaction between the gene therapy vector and a wild-type lentivirus obtained through natural infection. Upon co-infection of the same cell, RNA heterodimerization and co-packaging, chimeric retroviruses with unknown pathogenic potential may be generated by recombination. We already mentioned that the likelihood of recombination between the genomes of different retrovirus species is minute. Safety concerns are more serious in situations where the same retrovirus is encountered as gene therapy vector and as infectious agent. In this case, vector mobilization and recombination are a much more likely scenario (Bukovsky et al., 1999
). Extensive studies with animal retroviruses provide ample support for this scenario (DiFronzo & Holland, 1993
; Mikkelsen et al., 1996
; Schwartzberg et al., 1985
).
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balakrishnan, M., Fay, P. J. & Bambara, R. A. (2001). The kissing hairpin sequence promotes recombination within the HIV-1 5' leader region. Journal of Biological Chemistry 276, 36482-36492.
Beerens, N., Groot, F. & Berkhout, B. (2001). Initiation of HIV-1 reverse transcription is regulated by a primer activation signal. Journal of Biological Chemistry 276, 31247-31256.
Bender, W., Chien, Y. H., Chattopadhyay, S., Vogt, P. K., Gardner, M. B. & Davidson, N. (1978). High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures. Journal of Virology 25, 888-896.[Medline]
Berkhout, B. (1996). Structure and function of the human immunodeficiency virus leader RNA. Progress in Nucleic Acid Research and Molecular Biology 54, 1-34.[Medline]
Berkhout, B. & van Wamel, J. L. (1996). Role of the DIS hairpin in replication of human immunodeficiency virus type 1. Journal of Virology 70, 6723-6732.[Abstract]
Berkhout, B., Gatignol, A., Silver, J. & Jeang, K. T. (1990). Efficient trans-activation by the HIV-2 Tat protein requires a duplicated TAR RNA structure. Nucleic Acids Research 18, 1839-1846.[Abstract]
Berkhout, B., Essink, B. B. & Schoneveld, I. (1993). In vitro dimerization of HIV-2 leader RNA in the absence of PuGGAPuA motifs. FASEB Journal 7, 181-187.
Bobkov, A., Kazennova, E., Selimova, L., Bobkova, M., Khanina, T., Ladnaya, N., Kravchenko, A., Pokrovsky, V., Cheingsong-Popov, R. & Weber, J. (1998). A sudden epidemic of HIV type 1 among injecting drug users in the former Soviet Union: identification of subtype A, subtype B, and novel gagA/envB recombinants. AIDS Research and Human Retroviruses 14, 669-676.[Medline]
Bukovsky, A. A., Song, J. P. & Naldini, L. (1999). Interaction of human immunodeficiency virus-derived vectors with wild-type virus in transduced cells. Journal of Virology 73, 7087-7092.
Carr, J. K., Salminen, M. O., Koch, C., Gotte, D., Artenstein, A. W., Hegerich, P. A., St Louis, D., Burke, D. S. & McCutchan, F. E. (1996). Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand. Journal of Virology 70, 5935-5943.[Abstract]
Carr, J. K., Salminen, M. O., Albert, J., Sanders-Buell, E., Gotte, D., Birx, D. L. & McCutchan, F. E. (1998). Full genome sequences of human immunodeficiency virus type 1 subtypes G and A/G intersubtype recombinants. Virology 247, 22-31.[Medline]
Clever, J. L. & Parslow, T. G. (1997). Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation. Journal of Virology 71, 3407-3414.[Abstract]
Clever, J. L., Wong, M. L. & Parslow, T. G. (1996). Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA. Journal of Virology 70, 5902-5908.[Abstract]
Coffin, J. M. (1990). Retroviridae and their replication. In Fields Virology, 2nd edn. Edited by B. N. Fields & D. M. Knipe. New York: Raven Press.
Dardel, F., Marquet, R., Ehresmann, C., Ehresmann, B. & Blanquet, S. (1998). Solution studies of the dimerization initiation site of HIV-1 genomic RNA. Nucleic Acids Research 26, 3567-3571.
Das, A. T., Klaver, B., Klasens, B. I., van Wamel, J. L. & Berkhout, B. (1997). A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication. Journal of Virology 71, 2346-2356.[Abstract]
DiFronzo, N. L. & Holland, C. A. (1993). A direct demonstration of recombination between an injected virus and endogenous viral sequences, resulting in the generation of mink cell focus-inducing viruses in AKR mice. Journal of Virology 67, 3763-3770.[Abstract]
Dillon, P. J., Nelbock, P., Perkins, A. & Rosen, C. A. (1990). Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA. Journal of Virology 64, 4428-4437.[Medline]
Dirac, A. M., Huthoff, H., Kjems, J. & Berkhout, B. (2001). The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus type 2 RNA genome. Journal of Biological Chemistry 276, 32345-32352.
Dirac, A. M., Huthoff, H., Kjems, J. & Berkhout, B. (2002). Regulated HIV-2 RNA dimerization by means of alternative RNA conformations. Nucleic Acids Research 30, 2647-2655.
Emerman, M., Guyader, M., Montagnier, L., Baltimore, D. & Muesing, M. A. (1987). The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1. EMBO Journal 6, 3755-3760.[Abstract]
Evans, L. A., Moreau, J., Odehouri, K., Seto, D., Thomson-Honnebier, G., Legg, H., Barboza, A., Cheng-Mayer, C. & Levy, J. A. (1988). Simultaneous isolation of HIV-1 and HIV-2 from an AIDS patient. Lancet ii, 13891391.
Gao, F., Yue, L., Robertson, D. L., Hill, S. C., Hui, H., Biggar, R. J., Neequaye, A. E., Whelan, T. M., Ho, D. D., Shaw, G. M. and others (1994). Genetic diversity of human immunodeficiency virus type 2: evidence for distinct sequence subtypes with differences in virus biology. Journal of Virology 68, 74337447.[Abstract]
George, J. R., Ou, C. Y., Parekh, B., Brattegaard, K., Brown, V., Boateng, E. & De Cock, K. M. (1992). Prevalence of HIV-1 and HIV-2 mixed infections in Cote d'Ivoire. Lancet 340, 337-339.[Medline]
Girard, F., Barbault, F., Gouyette, C., Huynh-Dinh, T., Paoletti, J. & Lancelot, G. (1999). Dimer initiation sequence of HIV-1Lai genomic RNA: NMR solution structure of the extended duplex. Journal of Biomolecular Structure and Dynamics 16, 1145-1157.[Medline]
Gotte, M., Li, X. & Wainberg, M. A. (1999). HIV-1 reverse transcription: a brief overview focused on structurefunction relationships among molecules involved in initiation of the reaction. Archives of Biochemistry and Biophysics 365, 199-210.[Medline]
Greatorex, J. & Lever, A. (1998). Retroviral RNA dimer linkage. Journal of General Virology 79, 2877-2882.
Greatorex, J. S., Laisse, V., Dockhelar, M. C. & Lever, A. M. (1996). Sequences involved in the dimerisation of human T cell leukaemia virus type-1 RNA. Nucleic Acids Research 24, 2919-2923.
Haddrick, M., Lear, A. L., Cann, A. J. & Heaphy, S. (1996). Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1. Journal of Molecular Biology 259, 58-68.[Medline]
Hoglund, S., Ohagen, A., Goncalves, J., Panganiban, A. T. & Gabuzda, D. (1997). Ultrastructure of HIV-1 genomic RNA. Virology 233, 271-279.[Medline]
Huthoff, H. & Berkhout, B. (2001). Two alternating structures of the HIV-1 leader RNA. RNA 7, 143-157.
Jossinet, F., Lodmell, J. S., Ehresmann, C., Ehresmann, B. & Marquet, R. (2001). Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1. Journal of Biological Chemistry 276, 5598-5604.
Kaye, J. F. & Lever, A. M. (1998). Nonreciprocal packaging of human immunodeficiency virus type 1 and type 2 RNA: a possible role for the p2 domain of Gag in RNA encapsidation. Journal of Virology 72, 5877-5885.
Laughrea, M. & Jette, L. (1994). A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA. Biochemistry 33, 13464-13474.[Medline]
Laughrea, M. & Jette, L. (1996). Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248271 are dispensable for dimer formation. Biochemistry 35, 1589-1598.[Medline]
Laughrea, M., Jette, L., Mak, J., Kleiman, L., Liang, C. & Wainberg, M. A. (1997). Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization. Journal of Virology 71, 3397-3406.[Abstract]
Lever, A. M. (2000). HIV RNA packaging and lentivirus-based vectors. Advances in Pharmacology 48, 1-28.[Medline]
Lodmell, J. S., Ehresmann, C., Ehresmann, B. & Marquet, R. (2000). Convergence of natural and artificial evolution on an RNA looploop interaction: the HIV-1 dimerization initiation site. RNA 6, 1267-1276.
Lund, A. H., Mikkelsen, J. G., Schmidt, J., Duch, M. & Pedersen, F. S. (1999). The kissing-loop motif is a preferred site of 5' leader recombination during replication of SL3-3 murine leukemia viruses in mice. Journal of Virology 73, 9614-9618.
McBride, M. S. & Panganiban, A. T. (1996). The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. Journal of Virology 70, 2963-2973.[Abstract]
Malim, M. H., Bohnlein, S., Fenrick, R., Le, S. Y., Maizel, J. V. & Cullen, B. R. (1989). Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species. Proceedings of the National Academy of Sciences, USA 86, 8222-8226.[Abstract]
Marquet, R., Baudin, F., Gabus, C., Darlix, J. L., Mougel, M., Ehresmann, C. & Ehresmann, B. (1991). Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism. Nucleic Acids Research 19, 2349-2357.[Abstract]
Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology 288, 911-940.[Medline]
Mikkelsen, J. G., Lund, A. H., Kristensen, K. D., Duch, M., Sorensen, M. S., Jorgensen, P. & Pedersen, F. S. (1996). A preferred region for recombinational patch repair in the 5' untranslated region of primer binding site-impaired murine leukemia virus vectors. Journal of Virology 70, 1439-1447.[Abstract]
Mujeeb, A., Clever, J. L., Billeci, T. M., James, T. L. & Parslow, T. G. (1998). Structure of the dimer initiation complex of HIV-1 genomic RNA. Nature Structural Biology 5, 432-436.[Medline]
Mujeeb, A., Parslow, T. G., Zarrinpar, A., Das, C. & James, T. L. (1999). NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA. FEBS Letters 458, 387-392.[Medline]
Muriaux, D., Girard, P. M., Bonnet-Mathoniere, B. & Paoletti, J. (1995). Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5' leader region is evidenced by an antisense oligonucleotide. Journal of Biological Chemistry 270, 8209-8216.
Muriaux, D., De Rocquigny, H., Roques, B. P. & Paoletti, J. (1996a). NCp7 activates HIV-1Lai RNA dimerization by converting a transient looploop complex into a stable dimer. Journal of Biological Chemistry 271, 33686-33692.
Muriaux, D., Fosse, P. & Paoletti, J. (1996b). A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro. Biochemistry 35, 5075-5082.[Medline]
Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M. & Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267.[Abstract]
Negroni, M. & Buc, H. (2001). Retroviral recombination: what drives the switch? Nature Reviews in Molecular and Cellular Biology 2, 151-155.
Oude Essink, B. B., Das, A. T. & Berkhout, B. (1996). HIV-1 reverse transcriptase discriminates against non-self tRNA primers. Journal of Molecular Biology 264, 243-254.[Medline]
Paillart, J. C., Marquet, R., Skripkin, E., Ehresmann, B. & Ehresmann, C. (1994). Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. Journal of Biological Chemistry 269, 27486-27493.
Paillart, J. C., Westhof, E., Ehresmann, C., Ehresmann, B. & Marquet, R. (1997). Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA. Journal of Molecular Biology 270, 36-49.[Medline]
Peeters, M., Gershy-Damet, G. M., Fransen, K., Koffi, K., Coulibaly, M., Delaporte, E., Piot, P. & van der Groen, G. (1992). Virological and polymerase chain reaction studies of HIV-1/HIV-2 dual infection in Cote dIvoire. Lancet 340, 339-340.[Medline]
Peeters, M., Liegeois, F., Torimiro, N., Bourgeois, A., Mpoudi, E., Vergne, L., Saman, E., Delaporte, E. & Saragosti, S. (1999). Characterization of a highly replicative intergroup M/O human immunodeficiency virus type 1 recombinant isolated from a Cameroonian patient. Journal of Virology 73, 7368-7375.
Piyasirisilp, S., McCutchan, F. E., Carr, J. K., Sanders-Buell, E., Liu, W., Chen, J., Wagner, R., Wolf, H., Shao, Y., Lai, S., Beyrer, C. & Yu, X. F. (2000). A recent outbreak of human immunodeficiency virus type 1 infection in southern China was initiated by two highly homogeneous, geographically separated strains, circulating recombinant form AE and a novel BC recombinant. Journal of Virology 74, 11286-11295.
Rizvi, T. A. & Panganiban, A. T. (1993). Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. Journal of Virology 67, 2681-2688.[Abstract]
Robertson, D. L., Sharp, P. M., McCutchan, F. E. & Hahn, B. H. (1995). Recombination in HIV-1. Nature 374, 124-126.[Medline]
Robertson, D. L., Anderson, J. P., Bradac, J. A., Carr, J. K., Foley, B., Funkhouser, R. K., Gao, F., Hahn, B. H., Kalish, M. L., Kuiken, C., Learn, G. H., Leitner, T., McCutchan, F., Osmanov, S., Peeters, M., Pieniazek, D., Salminen, M., Sharp, P. M., Wolinsky, S. & Korber, B. (2000). HIV-1 nomenclature proposal. Science 288, 55-56.[Medline]
St Louis, D. C., Gotte, D., Sanders-Buell, E., Ritchey, D. W., Salminen, M. O., Carr, J. K. & McCutchan, F. E. (1998). Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro. Journal of Virology 72, 3991-3998.
Sakuragi, J. I. & Panganiban, A. T. (1997). Human immunodeficiency virus type 1 RNA outside the primary encapsidation and dimer linkage region affects RNA dimer stability in vivo. Journal of Virology 71, 3250-3254.[Abstract]
Salminen, M. O., Carr, J. K., Robertson, D. L., Hegerich, P., Gotte, D., Koch, C., Sanders-Buell, E., Gao, F., Sharp, P. M., Hahn, B. H., Burke, D. S. & McCutchan, F. E. (1997). Evolution and probable transmission of intersubtype recombinant human immunodeficiency virus type 1 in a Zambian couple. Journal of Virology 71, 2647-2655.[Abstract]
Schwartzberg, P., Colicelli, J. & Goff, S. P. (1985). Recombination between a defective retrovirus and homologous sequences in host DNA: reversion by patch repair. Journal of Virology 53, 719-726.[Medline]
Skripkin, E., Paillart, J. C., Marquet, R., Ehresmann, B. & Ehresmann, C. (1994). Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proceedings of the National Academy of Sciences, USA 91, 4945-4949.[Abstract]
Sundquist, W. I. & Heaphy, S. (1993). Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proceedings of the National Academy of Sciences, USA 90, 3393-3397.[Abstract]
Takehisa, J., Zekeng, L., Ido, E., Yamaguchi-Kabata, Y., Mboudjeka, I., Harada, Y., Miura, T., Kaptu, L. & Hayami, M. (1999). Human immunodeficiency virus type 1 intergroup (M/O) recombination in Cameroon. Journal of Virology 73, 6810-6820.
White, S. M., Renda, M., Nam, N. Y., Klimatcheva, E., Zhu, Y., Fisk, J., Halterman, M., Rimel, B. J., Federoff, H., Pandya, S., Rosenblatt, J. D. & Planelles, V. (1999). Lentivirus vectors using human and simian immunodeficiency virus elements. Journal of Virology 73, 2832-2840.
Received 8 January 2002;
accepted 5 June 2002.