Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M500382200 on January 12, 2005

J. Biol. Chem., Vol. 280, Issue 12, 10920-10924, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/12/10920    most recent
M500382200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haché, G.
Right arrow Articles by Harris, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haché, G.
Right arrow Articles by Harris, R. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Retroviral Hypermutation Specificity of APOBEC3F and APOBEC3G Is Governed by the C-terminal DNA Cytosine Deaminase Domain*{boxs}

Guylaine Haché, Mark T. Liddament, and Reuben S. Harris{ddagger}

From the University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics and the Institute for Molecular Virology, Minneapolis, Minnesota 55455

Received for publication, January 11, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human proteins APOBEC3F and APOBEC3G restrict retroviral infection by deaminating cytosine residues in the first cDNA strand of a replicating virus. These proteins have two putative deaminase domains, and it is unclear whether one or both catalyze deamination, unlike their homologs, AID and APOBEC1, which are well characterized single domain deaminases. Here, we show that only the C-terminal cytosine deaminase domain of APOBEC3F and -3G governs retroviral hypermutation. A chimeric protein with the N-terminal cytosine deaminase domain from APOBEC3G and the C-terminal cytosine deaminase domain from APOBEC3F elicited a dinucleotide hypermutation preference nearly indistinguishable from that of APOBEC3F. This 5'-TC->TT mutational specificity was confirmed in a heterologous Escherichia coli-based mutation assay, in which the 5'-CC->CT dinucleotide hypermutation preference of APOBEC3G also mapped to the C-terminal deaminase domain. An N-terminal APOBEC3G deletion mutant displayed a preference indistinguishable from that of the full-length protein, and replacing the C-terminal deaminase domain of APOBEC3F with AID resulted in an AID-like mutational signature. Together, these data indicate that only the C-terminal domain of APOBEC3F and -3G dictates the retroviral minus strand 5'-TC and 5'-CC dinucleotide hypermutation preferences, respectively, leaving the N-terminal domain to perform other aspects of retroviral restriction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The infectivity of a number of diverse retroviruses can be restricted by the human cellular proteins APOBEC3F1 and APOBEC3G, including HIV-1, simian immunodeficiency virus, equine infectious anemia virus, and murine leukemia virus (1–7). For HIV-1 restriction, several distinct steps are required (8). First, APOBEC3F and -3G must escape inactivation by the viral infectivity factor, Vif, which targets them for proteosomal degradation (9–15). Escape may be facilitated by the fact that APOBEC3F can partially resist HIV-1 Vif (4, 6). Second, APOBEC3F and -3G can independently, and possibly coordinately, gain access to the assembling viral particle through an association with the nucleocapsid region of Gag and viral genomic RNA (16–22). Third, once the virus infects a new cell, APOBEC3F and -3G deaminate nascent retroviral cDNA cytosines to uracils (1–7). These lesions manifest as genomic strand guanine to adenine hypermutations and can occur at strikingly high frequencies. Despite similar mechanisms of action and high levels of amino acid identity, APOBEC3F and -3G elicit distinct minus strand mutational preferences, 5'-TC and -CC, respectively (5'-GA and -GG on the viral genomic strand). Clear examples of both types of hypermutation occur in patient-derived HIV-1 DNA sequences indicating that these proteins are at least some of the time able to escape Vif-mediated degradation in vivo (4, 23).

Human APOBEC protein family members have either one or two cytosine/cytidine deaminase domains, each defined by a conserved HXE-X23–28-CPX2–4C motif (8, 24–26). Based on the three-dimensional structures of bacterial and yeast cytidine deaminases, the histidine and the cysteine residues are proposed to coordinate a zinc ion, whereas the glutamate promotes the creation of a hydroxide ion critical for deamination. The apoB mRNA editing protein APOBEC1 and the antibody gene deaminase AID are examples of single domain cytosine deaminases, and their activity is abolished by the mutation of the conserved deaminase domain residues (8, 26–28). In contrast, APOBEC3F and -3G have two deaminase domains, both of which appear to be essential for retroviral restriction (1, 3, 29). However, it is not presently clear whether one or both of these domains participates directly in the deamination reaction itself.

Here, we took advantage of the distinct dinucleotide hypermutation preferences of APOBEC3F and -3G (5'-TC and -CC, respectively) to show that this preference and the associated cytosine deaminase activity track exclusively with the C-terminal deaminase domain. The N-terminal deaminase domain was not required for C->U conversion, and it is likely important for other aspects of this innate retroviral restriction mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructions—Escherichia coli expression plasmids were derived from pTrc99A (AP BioSciences). The APOBEC3G expression construct was made by amplification of the human APOBEC3G cDNA from pRH204 (30) using oligonucleotides 431 and 365 (5'-NNG AGC TCA GGT ACC ACC ATG AAG CCT CAC TTC AGA AAC-3' and 5'-NNN NGT CGA CCC CAT CCT TCA GTT TTC CTG-3') and digestion of the PCR product with KpnI and SalI followed by ligation into pTrc99A. The human APOBEC3F expression plasmid was described previously (4), and this insert was flanked by identical restriction sites. The A3F+G and A3G+F chimeras were constructed by restricting the aforementioned plasmids with BsrG1, which cuts each APOBEC cDNA internally (Fig. 1A), and with either KpnI or SalI. Ligation of these plasmid backbones with the corresponding inserts produced A3F(1–162)+A3G(168–384) and A3G(1–167)+A3F(163–373), respectively. The A3F+AID expression plasmid was constructed by PCR amplification of the N-terminal region of APOBEC3F using oligonucleotides 431 and 459 (5'-G TAA AGA AAC TTC CTC CGG TTC ATT GCC TCC ATC GGG-3') and PCR amplification of AID using oligonucleotides 369 and 245 (5'-NN NNC CAT GGA CAG CCT CTT GAT GAA-3' and 5'-NNG GAT CCT GCA GTC AAA GTC CCA AAG TAC GAA ATG-3'), gel purification of the resulting products, followed by a second PCR amplification primed by complementary sequences within each product, and finally a third PCR amplification of the full-length chimeric cDNA using the 5'-APOBEC3F oligonucleotide 431 and the 3'-AID oligonucleotide 245. This product was digested with KpnI and BamHI and ligated into pTrc99A. Eukaryotic expression plasmids were derived from pcDNA3.1 (Invitrogen) by inserting KpnI and SalI-flanked APOBEC cDNAs from the E. coli expression plasmids. Qiagen DNA purification kits were used for small (mini)- and large (maxi)-scale plasmid purifications.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1.
Antiretroviral activity of chimeric APOBEC3F and APOBEC3G proteins. A, a schematic of APOBEC3F (A3F) and -3G (A3G) that depicts the site of recombination. The relative positions of the conserved N- and C-terminal cytosine deaminase domains are indicated by asterisks. aa, amino acids. B, infectivity of HIV-GFP produced in the presence of APOBEC3F (A3F), -3G (A3G), -3F+G (A3F+G), -3G+F (A3G+F), or a control vector (Vector). Results from two independent experiments were averaged and then normalized to the frequency of infection observed using viruses produced in the absence of any APOBEC protein (3.9 and 6.0% GFP-positive). Error bars report the difference in infectivity observed between the two experiments. C, immunoblots of virion-associated proteins using antibodies specific to the C terminus of APOBEC3G (upper panel) and to viral p24Gag (lower panel). APOBEC3G and APOBEC3F+G are predicted to be 384 and 379 amino acids, respectively, which explains their slightly different mobility. D, summary of the base substitution mutations found in HIV-GFP, which was produced in the presence of APOBEC3F, -3G, or -3G+F (gray, white, and black circles, respectively). G->A hypermutations and other base substitutions are depicted above and below the consensus sequence, respectively. Mutations from the indicated base to A, C, G, and T are illustrated by circles, squares, triangles, and diamonds, respectively. The consensus sequence is numbered based on the GFP gene open reading frame.

 
Single Cycle Infectivity Assays—293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Gemini Bioproducts), penicillin, and streptomycin (Invitrogen). HIV-GFP (also called CS-CG (31)) was produced by FuGENE 6 (Roche Applied Sciences)-mediated transfection of 50–70% confluent 293T cells with a plasmid mixture containing 0.22 µg of CS-CG, 0.14 µg of pRK5/Pack1(Gag-Pol), 0.07 µg of pRK5/Rev, 0.07 µg of pMDG (VSV-G Env), and 0.5 µg of an APOBEC expression or empty vector control plasmid as described previously (4). After an incubation period of 48 h, virus-containing supernatants were clarified by low speed centrifugation, filtered (0.45 µm), and quantified using a reverse transcriptase activitybased enzyme-linked immunosorbent assay (Cavidi Tech). Reverse transcriptase-normalized supernatants were applied to fresh 293T cells, and infection was allowed to proceed for 96 h. Infectivity (GFP fluorescence) was then measured by flow cytometry (FACSCalibur, BD Biosciences). For experiments requiring the recovery of retroviral DNA for hypermutation analyses, the viral supernatants were treated with 50 units/ml DNase (Sigma) prior to 293T cell infection.

Retroviral DNA Sequence Analyses—Genomic DNA was prepared from infected 293T cells using the DNeasy procedure (Qiagen) and incubated with DpnI to remove any non-reverse-transcribed CS-CG plasmid DNA. GPF was amplified using high fidelity PCR reagents (MJ Research) and CS-CG-specific primers 439 and 440 (5'-CG TGT ACG GTG GGA GGT CTA-3' and 5'-TT GGT AGC TGC TGT GTT GCT-3'). PCR products were cloned into pBluescript (Stratagene) as described previously (4) and sequenched using universal primers (University of Minnesota Advanced Genetic Analysis Center). Sequence analyses were done using Sequencher software (Gene Codes Corp.).

Virion Incorporation Assays—Viruses present in cell supernatants were harvested by filtration (0.45 µm), normalized for reverse transcriptase activity as measured by enzyme-linked immunosorbent assay (Cavidi Tech), and concentrated by ultracentrifugation through a 20% sucrose cushion (Beckman SW 41 rotor, 25,000 rpm, 2 h). Pellets were dissolved in SDS loading buffer and analyzed by Western blotting. APOBEC3G was detected with a rabbit anti-APOBEC3G, C terminus-specific antibody (32) from J. Lingappa (University of Washington). p24Gag was detected using a monoclonal antibody present in 183-H12-5C hybridoma serum. This hybridoma, originally from Drs. B. Chesebro and H. Chen, was obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (catalog no. 3537).

E. coli Mutation Assays—Uracil DNA glycosylase-deficient E. coli strain BW310 was transformed with isopropyl 1-thio-{beta}-D-galactopyranoside-inducible APOBEC expression constructs (above) or pTrc99A. Individual colonies were picked and grown to saturation in a rich medium containing 100 µg/ml ampicillin and 1 mM isopropyl 1-thio-{beta}-D-galactopyranoside. Appropriate dilutions were plated onto a rich medium containing 100 µg/ml rifampicin or ampicillin to select Rif R colonies or measure cell viability, respectively, after an overnight incubation. Mutation frequencies were reported as the number of Rif R colonies per 107 viable cells. rpoB sequences were obtained by colony PCR (oligonucleotides 441 and 442, 5'-TTG GCG AAA TGG CGG AAA ACC-3' and 5'-C ACC GAC GGA TAC CAC CTG CTG-3'), Exo-SAP (USB) treatment of the PCR product, and direct DNA sequencing (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To address whether the conserved N-terminal, C-terminal, or both cytosine deaminase domains of APOBEC3F and -3G are responsible for the observed retroviral dinucleotide hypermutation preferences, chimeric proteins were constructed by recombining the cDNA sequences at a common BsrG1 restriction site (Fig. 1A). The resulting protein fusion junction occurs within a conserved amino acid motif (FVYS), which is predicted to lie in a region linking the two deaminase domains (25). The resulting chimeric proteins were designated A3F+G and A3G+F. Should the strong dinucleotide preference of APOBEC3F or -3G be attributable to a single cytosine deaminase domain, then the dinucleotide preference would be expected to track with that domain in the corresponding chimera.

In comparison with HIV-GFP viruses produced in the presence of a control plasmid, retroviruses produced in the presence of APOBEC3F, -3G, or -3G+F expression constructs showed much lower levels of infectivity (Fig. 1B). In agreement with our previous studies (4), APOBEC3F was less inhibitory than APOBEC3G, causing a 40- versus 200-fold decrease in infectivity, respectively. The A3G+F chimera decreased retroviral infectivity nearly 10-fold. This effect was smaller than that attributable to equivalent amounts of APOBEC3F or -3G, but this slight decline in chimera efficacy is perhaps not surprising given that these proteins are at least several million years diverged (chimpanzees also express both APOBEC3F and -3G (8, 24, 33, 34)).

In contrast to the A3G+F chimera, expression of the reciprocal A3F+G hybrid protein failed to diminish the infectivity of HIV-GFP (Fig. 1B). To address whether this was due to an expression and/or incorporation deficiency, we examined the protein composition of viral particles produced in the presence of A3F+G, APOBEC3G, or a control vector after purification through a 20% sucrose cushion by ultra-centrifugation. Immunoblots of virion-associated proteins using a polyclonal antibody specific to the C terminus of APOBEC3G (32) showed that the A3F+G chimeric protein was encapsidated nearly as well as APOBEC3G (Fig. 1C). Levels of p24Gag were similar in all virion preparations. Thus, the apparent lack of chimera antiretroviral activity was not attributable to an expression or incorporation deficiency. The intrinsic deamination capacity of this chimeric protein will be addressed below.

The functional A3G+F chimera provided a molecular probe with which we could begin to delineate the deaminase domain(s) responsible for the distinct dinucleotide hypermutation preference of APOBEC3F and -3G. DNA was purified from 293T target cells infected with HIV-GFP viruses produced in the presence of APOBEC3F, -3G, -3G+F, or an empty control vector. Virus-specific GFP DNA sequences were amplified by high fidelity PCR, cloned, and sequenced. Like retroviruses exposed to APOBEC3F and -3G, those produced in the presence of the A3G+F chimera showed high levels of G->A hypermutation (Fig. 1D). As expected, the hypermutation frequencies correlated strongly with the observed infectivity declines, with APOBEC3G, -3F, and 3G+F triggering, respectively, 208, 82, and 56 G->A transition mutations per 10 kbp of viral DNA sequenced. The contribution from reverse transcription and PCR error was negligible inasmuch as only one base substitution (G474->A) was observed in more than 10 kbp of viral DNA sequences recovered in parallel from the control infections.

The mutated positions attributable to the A3G+F chimera were nearly identical to those of APOBEC3F (Fig. 1D). Prominent examples include G339, G363, G378, G474, G477, G492, and G642 at which over a quarter of the A3G+F and APOBECF hypermutations mapped. Overall, the genomic strand 5'-GA dinucleotide was preferred ~60% of the time by both A3G+F and APOBEC3F. In contrast, APOBEC3G displayed a strong preference for 5'-GG and rarely triggered mutations at 5'-GA, as observed previously (1–4, 35). These data are most consistent with the C-terminal cytosine deaminase domain of APOBEC3F (and by deduction APOBEC3G) being responsible for the retroviral dinucleotide hypermutation preferences observed.

To support these data, the mutational capacities and preferences of the chimeric A3F+G and A3G+F proteins were evaluated in an E. coli-based rifampicin-resistance (Rif R) mutation assay (4, 30, 36). This heterologous system provides a sensitive means to monitor the intrinsic DNA cytosine deaminase activity of the APOBEC proteins without possible complicating effects of other human and viral components. Bacteria expressing APOBEC3F and -3G were shown previously to display elevated Rif R mutation frequencies attributable to deamination within 5'-TC and -CC dinucleotide sequences (5'-GA and -GG on the complementary DNA strand), respectively (4, 30). Similar effects were observed here with APOBEC3F and -3G expression triggering 3.4- and 7.1-fold increases in the median mutation frequency (Fig. 2A). To our surprise, not only did expression of the A3G+F chimera cause a 3.5-fold increase in the median frequency of RifR, but expression of the corresponding A3F+G chimera triggered an even larger 4.6-fold increase. This stimulation suggested that the latter chimera was indeed capable of DNA cytosine deamination, further permitting a two-way evaluation of the intrinsic mutational preferences of these hybrid proteins.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 2.
RifR mutation profile of APOBEC proteins expressed in E. coli. A, observed mutation frequencies for 12 independent E. coli cultures expressing APOBEC3F (A3F), -3G (A3G), -3F+G (A3F+G), -3G+F (A3G+F), or a control vector (Vector). Each data point corresponds to the mutation frequency obtained using a single culture. The median mutation frequency is shown for each condition. B, a histogram showing the rpoB gene mutational preferences for APOBEC3F and -3G+F(e.g. C1535, G1546, and C1721) and for APOBEC3G and -3F+G(e.g. C1691). Only the well mutated cytosines or guanines (C on the opposite strand) within rpoB are indicated. 95, 61, 22, 31, and 20 independent RifR mutants were sequenced from cells expressing the vector control, APOBEC3G, -3F, -3F+G, and -3G+F, respectively. Raw sequencing data are shown in supplemental Fig. 1.

 
A limited number of C/G->T/A base substitutions within a 0.2-kbp interval of the E. coli RNA polymerase B (rpoB) gene confer Rif R. Mutable cytosines can be found on either DNA stand and within all possible dinucleotide contexts. Independent RifR mutants were therefore purified and subjected to PCR and DNA sequencing to determine the base changes responsible for the drug-resistant phenotype. The main rpoB hotspots preferred by the A3G+F chimera were nearly identical to those preferred by APOBEC3F (C1721 > C1535 > G1546) and were occurring at levels above those found in the control vector containing cells (Fig. 2B and supplemental Fig. 1). These sites harbored the majority of all Rif R mutations attributable to these proteins, and for APOBEC3F these data agreed with those from our previous studies (4). Such a correlation was even more profound for the A3F+G chimera that, like APOBEC3G (30), almost exclusively preferred the last cytosine of a 5'-CCCC run (C1691, Fig. 2B). This indicated that the A3F+G chimera was fully capable of DNA cytosine deamination. Together with the fact that the A3F+G chimera also incorporated into retroviral particles (Fig. 1C), these data suggested that a novel step between incorporation and cytosine deamination was disrupted, an intriguing possibility that we have begun to pursue. Moreover, these data strengthened the conclusion that the C-terminal cytosine deaminase domain alone dictates (and presumably also catalyzes) the dinucleotide hypermutation preferences of both APOBEC3F and -3G.

However, it was still formally possible that the C-terminal cytosine deaminase domain of APOBEC3F and -3G somehow guides the N-terminal cytosine deaminase domain to the specific DNA target (i.e. selects the appropriate dinucleotide for deamination by the N-terminal deaminase domain). We therefore employed two approaches to address this possibility and distinguish it from the more likely aforementioned alternative. First, we examined the mutational capacity and specificity of an APOBEC3G mutant lacking most of its N-terminal deaminase domain (A3G{Delta}N(1–67)). This deletion removed the catalytic HXE motif of the N-terminal domain. E. coli expressing this mutant showed levels and distributions of Rif R mutations indistinguishable from those attributable to expression of the full-length protein (predominantly C/G->T/A transitions at C1691; data not shown). This result demonstrated that the N-terminal deaminase domain is not required for catalysis. It is notable that a similar APOBEC3G N-terminal deletion mutant fails to restrict retroviral infectivity (37).

Second, if the C-terminal deaminase domain alone catalyzes the lesions that result in hypermutations, then the replacement of this domain with that of another bona fide DNA cytosine deaminase should yield a chimera with the dinucleotide specificity of the latter protein. We chose to replace the C-terminal domain of APOBEC3F with AID, a single domain APOBEC protein family member that uses cytosine deamination to trigger antibody gene diversification events in vertebrates (27, 28) (Fig. 3A). Bacterial expression of the resulting A3F+AID chimera caused a modest 2–3-fold increase in the frequency of Rif R mutants (Fig. 3B). However, an examination of the mutated rpoB genes revealed a striking change in the distribution of the mutations that confer Rif R (Fig. 3C and supplemental Fig. 1). The A3F+AID chimera produced an AID-like mutational specificity (36), with the majority of the C/G->T/A transition mutations occurring predominantly at two positions within rpoB, G1586 > C1576. These preferences hardly overlap with those of APOBEC3F or its derivatives (Fig. 2B and supplemental Fig. 1), indicating that the mutational specificity of the C-terminal deaminase domain of APOBEC3F can be transformed completely by replacing it with the single domain deaminase AID.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 3.
An APOBEC3F-AID chimera elicits an AID-like mutation pattern. A, schematic of the construction of the A3F+AID chimera. The relative positions of the conserved cytosine deaminase domains are indicated by asterisks. B and C, the observed Rif R mutation frequencies (n = 8) and distributions for E. coli expressing a control vector, AID, or A3F+AID. 93 and 37 independent RifR mutants were sequenced from cells expressing AID or A3F+AID, respectively. The vector rpoB mutation data are identical to those shown in Fig. 2C and are illustrated again here for comparison. The experimental parameters are identical to those described in Fig. 2. Raw sequencing data are shown in supplemental Fig. 1. aa, amino acid(s).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mutation specificities of APOBEC family member chimeras and deletion mutants demonstrate that the C-terminal cytosine deaminase domain of APOBEC3F and -3G is exclusively responsible for the retroviral dinucleotide hypermutation preference and for catalysis. Respectively, these proteins prefer to deaminate cytosines within 5'-TC and 5'-CC on the retroviral cDNA strand, alterations that manifest as genomic strand 5'-GA->AA and 5'-GG->AG hypermutations. Such biases are readily visible in model systems and in patient-derived HIV-1 sequences. The delineation of the active cytosine deaminase domain is an important step toward fully understanding the molecular details of this mechanism of retroviral restriction.

Previous APOBEC3G site-directed mutation experiments showed that both the N- and the C-terminal cytosine deaminase domains are required for inhibiting the infectivity of HIV-based retroviruses (1, 3). These studies used APOBEC3G variants with mutations in the conserved cytosine deaminase domain cysteine and glutamate residues thought to be crucial for zinc-binding and catalysis, respectively. Both studies reported that mutations in either deaminase domain rendered APOBEC3G unable to restrict infection, and it was therefore not possible to define the source of the deaminase activity causing retroviral hypermutation or whether other mechanistic steps were disrupted. In contrast, a third study indicated that the N-terminal glutamate was dispensable, whereas the C-terminal glutamate of APOBEC3G was essential for restricting HIV-1 infection (29). Although this initially appears consistent with our data, both mutants still showed moderate levels of G->A mutation suggesting that either domain can mediate catalysis (29). This apparent conundrum may be attributable to reverse transcriptase error and/or to the APOBEC3G mutants retaining a low level of deaminase activity.

Here, functional APOBEC chimeras and deletion mutants were used to unambiguously show that, for both APOBEC3F and APOBEC3G, the C-terminal cytosine deaminase domain alone governs the catalytic activity and the retroviral hypermutation specificity. The N-terminal deaminase domain is also important for retroviral restriction, but it is likely required for mechanistic steps preceding deamination (e.g. virion incorporation (17)). It is probable that during the evolution of this unique innate antiretroviral immune response, two single domain deaminases recombined to yield a dual domain deaminase with a target tropism and mutator activity that eventually were governed by the N- and C-terminal domains, respectively.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

{ddagger} A 2004 Searle Scholar and supported by a Burroughs-Wellcome Fund Hitchings-Elion fellowship and a new laboratory start-up award from the University of Minnesota. To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 321 Church St. S.E., 6-155 Jackson Hall, Minneapolis, MN 55455. Tel.: 612-624-0457; Fax: 612-625-2163; E-mail: rsh{at}umn.edu.

1 The abbreviations used are: APOBEC, apolipoprotein B editing catalytic; AID, activation-induced deaminase; HIV-1, human immunodeficiency virus 1; GFP, green fluorescent protein; Rif R, rifampicin resistance. Back


   ACKNOWLEDGMENTS
 
We thank S. Jonsson for help with data analysis; W. Brown, N. Jahren, D. Livingston, and A. Schumacher for helpful comments; and J. Lingappa and N. Somia for the generous provision of immunoreagents. The p24Gag hybridoma (183-H12-5C, catalog no. 3537, from Drs. B. Chesebro and H. Chen) was obtained through the National Institutes of Health AIDS Research and Reference Reagent Program.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mangeat, B., Turelli, P., Caron, G., Friedli, M., Perrin, L., and Trono, D. (2003) Nature 424, 99–103[CrossRef][Medline] [Order article via Infotrieve]
  2. Harris, R. S., Bishop, K. N., Sheehy, A. M., Craig, H. M., Petersen-Mahrt, S. K., Watt, I. N., Neuberger, M. S., and Malim, M. H. (2003) Cell 113, 803–809[CrossRef][Medline] [Order article via Infotrieve]
  3. Zhang, H., Yang, B., Pomerantz, R. J., Zhang, C., Arunachalam, S. C., and Gao, L. (2003) Nature 424, 94–98[CrossRef][Medline] [Order article via Infotrieve]
  4. Liddament, M. T., Brown, W. L., Schumacher, A. J., and Harris, R. S. (2004) Curr. Biol. 14, 1385–1391[CrossRef][Medline] [Order article via Infotrieve]
  5. Bishop, K. N., Holmes, R. K., Sheehy, A. M., Davidson, N. O., Cho, S. J., and Malim, M. H. (2004) Curr. Biol. 14, 1392–1396[CrossRef][Medline] [Order article via Infotrieve]
  6. Wiegand, H. L., Doehle, B. P., Bogerd, H. P., and Cullen, B. R. (2004) EMBO J. 23, 2451–2458[CrossRef][Medline] [Order article via Infotrieve]
  7. Zheng, Y. H., Irwin, D., Kurosu, T., Tokunaga, K., Sata, T., and Peterlin, B. M. (2004) J. Virol. 78, 6073–6076[Abstract/Free Full Text]
  8. Harris, R. S., and Liddament, M. T. (2004) Nat. Rev. Immunol. 4, 868–877[CrossRef][Medline] [Order article via Infotrieve]
  9. Conticello, S. G., Harris, R. S., and Neuberger, M. S. (2003) Curr. Biol. 13, 2009–2013[CrossRef][Medline] [Order article via Infotrieve]
  10. Mehle, A., Strack, B., Ancuta, P., Zhang, C., McPike, M., and Gabuzda, D. (2004) J. Biol. Chem. 279, 7792–7798[Abstract/Free Full Text]
  11. Yu, X., Yu, Y., Liu, B., Luo, K., Kong, W., Mao, P., and Yu, X. F. (2003) Science 302, 1056–1060[Abstract/Free Full Text]
  12. Marin, M., Rose, K. M., Kozak, S. L., and Kabat, D. (2003) Nat. Med. 9, 1398–1403[CrossRef][Medline] [Order article via Infotrieve]
  13. Sheehy, A. M., Gaddis, N. C., and Malim, M. H. (2003) Nat. Med. 9, 1404–1407[CrossRef][Medline] [Order article via Infotrieve]
  14. Stopak, K., de Noronha, C., Yonemoto, W., and Greene, W. C. (2003) Mol. Cell 12, 591–601[CrossRef][Medline] [Order article via Infotrieve]
  15. Mehle, A., Goncalves, J., Santa-Marta, M., McPike, M., and Gabuzda, D. (2004) Genes Dev. 18, 2861–2866[Abstract/Free Full Text]
  16. Alce, T. M., and Popik, W. (2004) J. Biol. Chem. 279, 34083–34086[Abstract/Free Full Text]
  17. Cen, S., Guo, F., Niu, M., Saadatmand, J., Deflassieux, J., and Kleiman, L. (2004) J. Biol. Chem. 279, 33177–33184[Abstract/Free Full Text]
  18. Svarovskaia, E. S., Xu, H., Mbisa, J. L., Barr, R., Gorelick, R. J., Ono, A., Freed, E. O., Hu, W. S., and Pathak, V. K. (2004) J. Biol. Chem. 279, 35822–35828[Abstract/Free Full Text]
  19. Douaisi, M., Dussart, S., Courcoul, M., Bessou, G., Vigne, R., and Decroly, E. (2004) Biochem. Biophys. Res. Commun. 321, 566–573[CrossRef][Medline] [Order article via Infotrieve]
  20. Zennou, V., Perez-Caballero, D., Gottlinger, H., and Bieniasz, P. D. (2004) J. Virol. 78, 12058–12061[Abstract/Free Full Text]
  21. Luo, K., Liu, B., Xiao, Z., Yu, Y., Yu, X., Gorelick, R., and Yu, X. F. (2004) J. Virol. 78, 11841–11852[Abstract/Free Full Text]
  22. Schafer, A., Bogerd, H. P., and Cullen, B. R. (2004) Virology 328, 163–168[CrossRef][Medline] [Order article via Infotrieve]
  23. Beale, R. C., Petersen-Mahrt, S. K., Watt, I. N., Harris, R. S., Rada, C., and Neuberger, M. S. (2004) J. Mol. Biol. 337, 585–596[CrossRef][Medline] [Order article via Infotrieve]
  24. Conticello, S. G., Thomas, C. J., Petersen-Mahrt, S., and Neuberger, M. S. (2005) Mol. Biol. Evol. 22, 367–377[Abstract/Free Full Text]
  25. Jarmuz, A., Chester, A., Bayliss, J., Gisbourne, J., Dunham, I., Scott, J., and Navaratnam, N. (2002) Genomics 79, 285–296[CrossRef][Medline] [Order article via Infotrieve]
  26. Wedekind, J. E., Dance, G. S., Sowden, M. P., and Smith, H. C. (2003) Trends Genet. 19, 207–216[CrossRef][Medline] [Order article via Infotrieve]
  27. Honjo, T., Muramatsu, M., and Fagarasan, S. (2004) Immunity 20, 659–668[CrossRef][Medline] [Order article via Infotrieve]
  28. Neuberger, M. S., Harris, R. S., Di Noia, J., and Petersen-Mahrt, S. K. (2003) Trends Biochem. Sci. 28, 305–312[CrossRef][Medline] [Order article via Infotrieve]
  29. Shindo, K., Takaori-Kondo, A., Kobayashi, M., Abudu, A., Fukunaga, K., and Uchiyama, T. (2003) J. Biol. Chem. 278, 44412–44416[Abstract/Free Full Text]
  30. Harris, R. S., Petersen-Mahrt, S. K., and Neuberger, M. S. (2002) Mol. Cell 10, 1247–1253[CrossRef][Medline] [Order article via Infotrieve]
  31. Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. H., and Verma, I. M. (1998) J. Virol. 72, 8150–8157[Abstract/Free Full Text]
  32. Newman, E. N. C., Holmes, R. K., Craig, H. M., Klein, K. C., Lingappa, J. R., Malim, M. H., and Sheehy, A. M. (2005) Curr. Biol. 15, 166–170[CrossRef][Medline] [Order article via Infotrieve]
  33. Zhang, J., and Webb, D. M. (2004) Hum. Mol. Genet. 13, 1785–1791[Abstract/Free Full Text]
  34. Sawyer, S. L., Emerman, M., and Malik, H. S. (2004) PLoS Biol. 2, E275[CrossRef][Medline] [Order article via Infotrieve]
  35. Yu, Q., Konig, R., Pillai, S., Chiles, K., Kearney, M., Palmer, S., Richman, D., Coffin, J. M., and Landau, N. R. (2004) Nat. Struct. Mol. Biol. 11, 435–442[CrossRef][Medline] [Order article via Infotrieve]
  36. Petersen-Mahrt, S. K., Harris, R. S., and Neuberger, M. S. (2002) Nature 418, 99–103[Medline] [Order article via Infotrieve]
  37. Li, J., Potash, M. J., and Volsky, D. J. (2004) J. Cell. Biochem. 92, 560–572[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
J. Zielonka, I. G. Bravo, D. Marino, E. Conrad, M. Perkovic, M. Battenberg, K. Cichutek, and C. Munk
Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases
J. Virol., August 1, 2009; 83(15): 7547 - 7559.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
S. Henriet, G. Mercenne, S. Bernacchi, J.-C. Paillart, and R. Marquet
Tumultuous Relationship between the Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) and the Human APOBEC-3G and APOBEC-3F Restriction Factors
Microbiol. Mol. Biol. Rev., June 1, 2009; 73(2): 211 - 232.
[Abstract] [Full Text] [PDF]

Home page
 Phil Trans R Soc BHome page
Y.-L. Chiu and W. C Greene
APOBEC3G: an intracellular centurion
Phil Trans R Soc B, March 12, 2009; 364(1517): 689 - 703.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. A. Russell, J. Smith, R. Barr, D. Bhattacharyya, and V. K. Pathak
Distinct Domains within APOBEC3G and APOBEC3F Interact with Separate Regions of Human Immunodeficiency Virus Type 1 Vif
J. Virol., February 15, 2009; 83(4): 1992 - 2003.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. P. Bogerd, R. L. Tallmadge, J. L. Oaks, S. Carpenter, and B. R. Cullen
Equine Infectious Anemia Virus Resists the Antiretroviral Activity of Equine APOBEC3 Proteins through a Packaging-Independent Mechanism
J. Virol., December 1, 2008; 82(23): 11889 - 11901.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. P. Bennett, J. D. Salter, X. Liu, J. E. Wedekind, and H. C. Smith
APOBEC3G Subunits Self-associate via the C-terminal Deaminase Domain
J. Biol. Chem., November 28, 2008; 283(48): 33329 - 33336.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
A. Arudchandran, R. M. Bernstein, and E. E. Max
Single-strand DNA breaks in Ig class switch recombination that depend on UNG but not AID
Int. Immunol., November 1, 2008; 20(11): 1381 - 1393.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. D. Stenglein, H. Matsuo, and R. S. Harris
Two Regions within the Amino-Terminal Half of APOBEC3G Cooperate To Determine Cytoplasmic Localization
J. Virol., October 1, 2008; 82(19): 9591 - 9599.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
H. P. Bogerd and B. R. Cullen
Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation
RNA, June 1, 2008; 14(6): 1228 - 1236.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Chelico, E. J. Sacho, D. A. Erie, and M. F. Goodman
A Model for Oligomeric Regulation of APOBEC3G Cytosine Deaminase-dependent Restriction of HIV
J. Biol. Chem., May 16, 2008; 283(20): 13780 - 13791.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M.-A. Langlois and M. S. Neuberger
Human APOBEC3G Can Restrict Retroviral Infection in Avian Cells and Acts Independently of both UNG and SMUG1
J. Virol., May 1, 2008; 82(9): 4660 - 4664.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
A. J. Schumacher, G. Hache, D. A. MacDuff, W. L. Brown, and R. S. Harris
The DNA Deaminase Activity of Human APOBEC3G Is Required for Ty1, MusD, and Human Immunodeficiency Virus Type 1 Restriction
J. Virol., March 15, 2008; 82(6): 2652 - 2660.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
E. P. Browne and D. R. Littman
Species-Specific Restriction of Apobec3-Mediated Hypermutation
J. Virol., February 1, 2008; 82(3): 1305 - 1313.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. L. Wiegand and B. R. Cullen
Inhibition of Alpharetrovirus Replication by a Range of Human APOBEC3 Proteins
J. Virol., December 15, 2007; 81(24): 13694 - 13699.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
M. Bonvin and J. Greeve
Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro
J. Gen. Virol., December 1, 2007; 88(12): 3270 - 3274.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, et al.
Human Immunodeficiency Virus Type 1 cDNAs Produced in the Presence of APOBEC3G Exhibit Defects in Plus-Strand DNA Transfer and Integration
J. Virol., July 1, 2007; 81(13): 7099 - 7110.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
H. Muckenfuss, J. K. Kaiser, E. Krebil, M. Battenberg, C. Schwer, K. Cichutek, C. Munk, and E. Flory
Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene
Nucleic Acids Res., June 28, 2007; 35(11): 3784 - 3796.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. K. Holmes, F. A. Koning, K. N. Bishop, and M. H. Malim
APOBEC3F Can Inhibit the Accumulation of HIV-1 Reverse Transcription Products in the Absence of Hypermutation: COMPARISONS WITH APOBEC3G
J. Biol. Chem., January 26, 2007; 282(4): 2587 - 2595.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Wang, P. T. Dolan, Y. Dang, and Y.-H. Zheng
Biochemical Differentiation of APOBEC3F and APOBEC3G Proteins Associated with HIV-1 Life Cycle
J. Biol. Chem., January 19, 2007; 282(3): 1585 - 1594.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
F. Guo, S. Cen, M. Niu, J. Saadatmand, and L. Kleiman
Inhibition of Formula-Primed Reverse Transcription by Human APOBEC3G during Human Immunodeficiency Virus Type 1 Replication
J. Virol., December 1, 2006; 80(23): 11710 - 11722.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
S. R. Jonsson, G. Hache, M. D. Stenglein, S. C. Fahrenkrug, V. Andresdottir, and R. S. Harris
Evolutionarily conserved and non-conserved retrovirus restriction activities of artiodactyl APOBEC3F proteins
Nucleic Acids Res., November 14, 2006; 34(19): 5683 - 5694.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
Y. Dang, X. Wang, W. J. Esselman, and Y.-H. Zheng
Identification of APOBEC3DE as Another Antiretroviral Factor from the Human APOBEC Family
J. Virol., November 1, 2006; 80(21): 10522 - 10533.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
K. N. Bishop, R. K. Holmes, and M. H. Malim
Antiviral Potency of APOBEC Proteins Does Not Correlate with Cytidine Deamination.
J. Virol., September 1, 2006; 80(17): 8450 - 8458.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. D. Stenglein and R. S. Harris
APOBEC3B and APOBEC3F Inhibit L1 Retrotransposition by a DNA Deamination-independent Mechanism
J. Biol. Chem., June 23, 2006; 281(25): 16837 - 16841.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. P. Bogerd, H. L. Wiegand, A. E. Hulme, J. L. Garcia-Perez, K. S. O'Shea, J. V. Moran, and B. R. Cullen
Cellular inhibitors of long interspersed element 1 and Alu retrotransposition
PNAS, June 6, 2006; 103(23): 8780 - 8785.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
Y. Iwatani, H. Takeuchi, K. Strebel, and J. G. Levin
Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect.
J. Virol., June 1, 2006; 80(12): 5992 - 6002.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. OhAinle, J. A. Kerns, H. S. Malik, and M. Emerman
Adaptive Evolution and Antiviral Activity of the Conserved Mammalian Cytidine Deaminase APOBEC3H.
J. Virol., April 1, 2006; 80(8): 3853 - 3862.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y.-L. Chiu and W. C. Greene
APOBEC3 Cytidine Deaminases: Distinct Antiviral Actions along the Retroviral Life Cycle
J. Biol. Chem., March 31, 2006; 281(13): 8309 - 8312.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
B. R. Cullen
Role and Mechanism of Action of the APOBEC3 Family of Antiretroviral Resistance Factors
J. Virol., February 1, 2006; 80(3): 1067 - 1076.
[Full Text] [PDF]

Home page
 J. Virol.Home page
F. Delebecque, R. Suspene, S. Calattini, N. Casartelli, A. Saib, A. Froment, S. Wain-Hobson, A. Gessain, J.-P. Vartanian, and O. Schwartz
Restriction of Foamy Viruses by APOBEC Cytidine Deaminases
J. Virol., January 15, 2006; 80(2): 605 - 614.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/12/10920    most recent
M500382200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haché, G.
Right arrow Articles by Harris, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haché, G.
Right arrow Articles by Harris, R. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement