Hypermutation of HIV-1 DNA in the Absence of the Vif Protein
Denise Lecossier,
Francine Bouchonnet,
François Clavel,
Allan J. Hance*
INSERM U552, Paris, France.
* To whom correspondence should be addressed. E-mail: hance@bichat.inserm.fr
The Vif (viral infectivity factor) protein of human immunodeficiency virus–1 (HIV-1) is essential for the production of infectious virus by T lymphocytes and macrophages, the natural targets for HIV-1 infection. Vif is dispensable for HIV infection in certain transformed cell lines ("permissive" cells) but is required for propagative infection in others ("nonpermissive" cells). Vif is thought to inhibit an antiviral pathway recently demonstrated to require the cellular protein CEM 15 (1). This protein, also called APOBEC3G (2), is a member of the family of cytidine deaminases. Because recent studies have suggested that some mammalian cytidine deaminases target single-stranded DNA (3), we tested the hypothesis that Vif may be required to prevent the editing of minus-strand viral DNA synthesized by virions produced in nonpermissive cells. Thus, nonpermissive H9 cells (4) were exposed to virus produced by HeLa cells transfected with wild-type HIV-1 (pNL4-3) or a variant in which a portion of the vif gene had been deleted ( vif). The virions produced by H9 cells 24 to 48 hours after infection were recovered. Target cells (CD4+, HeLa-derived P4 cells) were then exposed to these virions and incubated for 5 hours at 37°C, and newly reverse transcribed viral DNA was isolated (4). Degenerate primers that would not be affected by the editing of minus-strand DNA by cytidine deaminases were then used to amplify viral sequences in env and U5. The products were then cloned and sequenced. Guanine to adenine (G A) changes were significantly more frequent in DNA synthesized by vif virions than in DNA produced by wild-type virions (P < 0.0001 and P < 0.01) (Fig. 1A; fig. S1A and fig. S2). In contrast, no difference in the frequency of GA changes could be observed in viral DNA synthesized by wild-type and vif viruses produced in permissive cells (P4 cells) under the same conditions, or those generated in HeLa cells (fig. S1).
Fig. 1. Sequence analysis of env for viral DNA synthesized in infected P4 cells, cDNA prepared from viral RNA, and DNA synthesized in endogenous RT reactions. (A) DNA was isolated from P4 cells cultured for 5 hours after spinoculation with virions produced by H9 cells infected by cell-free virions produced by transfected HeLa cells. (B) cDNA synthesized by M-MLV RT using RNA from vif virions produced by H9 cells <24 hours after infection with virions produced by transfected HeLa cells. (C) Endogenous RT reactions performed using virions produced by H9 cells. The sequence of pNL4-3 for the amplified fragment is shown at the top of the figure (annotated per GenBank M19921
[GenBank]
), and only bases different from this sequence are shown for the other sequences. The corresponding amino acids for pNL4-3 are shown above the sequence; amino acid substitutions or premature stop codons (*) resulting from GA changes observed in the study are shown below the involved base. The numbers preceding the sequences (1x, 5x,...) indicate the number of clones with the given sequence. Only the portion of the sequence containing mutations is shown.
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GA changes in the HIV plus-strand DNA can be directly explained by modifications of cytidine to uridine (CU) in the minus-strand of DNA by a cytidine deaminase, because U is subsequently read as T by DNA polymerases. Such changes might also be compatible with hypermutation by reverse transcriptase, a phenomenon markedly accentuated by low intracellular deoxycytidine triphosphate to deoxythymidine triphosphate (dCTP/dTTP) ratios (5). Because the dCTP/dTTP ratio present in P4 cells is not conducive to hypermutation (6) and because base changes were only frequent when virions were produced by nonpermissive cells in the absence of Vif, this mechanism seems less likely. Similarly, the editing of viral RNA by a cytidine deaminase would not explain our findings, because CT changes and not GA changes in the DNA plus strand would then be expected. Indeed, when RNA was extracted from vif virions produced by H9 cells during the first 24 hours after infection, reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (M-MLV RT), and amplified, no GA changes could be observed in these cDNA sequences (Fig. 1B). However, when these virions were used to synthesize viral DNA by endogenous reverse transcription within the viral particles (4), numerous GA changes were seen (Fig. 1C). GA changes were not observed when endogenous reverse transcription was performed using wild-type viruses produced by H9 cells or vif viruses produced by permissive cells (Fig. 1C) (6).
Overall, our results indicate that in the absence of Vif, virions produced in nonpermissive H9 cells contain an activity that results in the introduction of C U changes into the minus-strand of viral DNA synthesized during reverse transcription, a feature that is highly compatible with editing of the minus-strand by a cytidine deaminase, such as CEM 15/APOBEC3G (1). Thus, when HIV replicates in cells expressing CEM 15/APOBEC3G, the Vif protein, through mechanisms that remain to be defined, is able to prevent the accumulation of multiple defects in structural, enzymatic, and regulatory viral proteins that would otherwise result in failure at several points in the HIV life cycle.
References and Notes
| 1. | A. M. Sheehy, N. C. Gaddis, J. D. Choi, M. H. Malim, Nature 418, 646 (2002).[CrossRef][ISI][Medline] |
| 2. | A. Jarmuz et al., Genomics 79, 285 (2002).[CrossRef][ISI][Medline] |
| 3. | A. Martin, M. D. Scharff, Proc. Natl. Acad Sci. U.S.A. 99, 12304 (2002).[Abstract/Free Full Text] |
| 4. | Materials and Methods are available as supporting online material at Science Online. |
| 5. | M. A. Martinez, J. P. Vartanian, S. Wain-Hobson, Proc. Natl. Acad. Sci. U.S.A. 91, 11787 (1994).[Abstract/Free Full Text] |
| 6. | A. J. Hance, unpublished data. |
Supporting Online Material
www.sciencemag.org/cgi/content/full/300/5622/1112/DC1
Materials and Methods
Figs. S1 and S2
10.1126/science.1083338
Include this information when citing this paper.
Volume 300,
Number 5622,
Issue of 16 May 2003,
p. 1112.
Copyright © 2003 by The American Association for the Advancement of Science. All rights reserved.
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