Testing the reverse transcriptase model of somatic mutation

Molecular Immunology 38 (2001) 303– 311 www.elsevier.com/locate/molimm

Testing the reverse transcriptase model of somatic mutation Stephen Z. Sack, Philip D. Bardwell, Matthew D. Scharff * Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park A6enue, Bronx, NY 10461, USA Received 5 March 2001; received in revised form 11 June 2001; accepted 12 June 2001

Abstract Somatic hypermutation of the variable (V) regions of rearranged immunoglobulin genes leads to antibody affinity maturation. Although this process has been extensively studied, the mechanisms responsible for these multiple point mutations are still elusive. One mechanism that was proposed over 10 years ago by Steele and Pollard was that an intrinsic reverse transcriptase (RT) copies the nascent mRNA creating the large number of observed point mutations due to its high error rate. A cDNA copy of the mutated V region would then replace the endogenous DNA through a gene conversion-like event, thus integrating these point mutations into the genome. This model of hypermutation would account for the very high mutation rate, the presence of hotspots, strand bias, the requirement for transcription and localization of mutation within the immunoglobulin V region. Using AZT and ddC to inhibit endogenous RTs, we have assayed for somatic mutation using a murine in vivo model. Somatic mutation occurred at similar frequencies and with the same characteristics with or without treatment of RT inhibitors, suggesting that standard reverse transcription is not required for antibody V region hypermutation in the mouse. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Generation of diversity; Antibodies; B lymphocytes; mRNA

1. Introduction In mice and humans, a large repertoire of antibodies with different antigen binding sites is created through the rearrangement of variable (V), diversity (D) and joining (J) region germline minigenes to form mature variable regions. After antigenic challenge and activation by T cells, the immunoglobulin gene undergoes a process of V region somatic hypermutation (Harris et al., 1999; Weigert et al., 1970). During this process, single point mutations occur at a high rate within the V region and its immediate flanking sequences and accumulate over time. Some of these mutations alter the affinity and binding specificity of the antibodies, and the B cells producing higher affinity antibodies are selected for in the germinal centers of peripheral lymphoid organs and go on to dominate the antibody response (Berek and Milstein, 1987; Kelsoe, 1996; Kocks and Rajewsky, 1985). * Corresponding author. Tel.: + 1-718-430-3527; fax: +1-718-4308574. E-mail address: [email protected] (M.D. Scharff).

The molecular mechanism of V region hypermutation remains elusive, even though the characteristics of the process are well known (Harris et al., 1999; Storb, 1998). Most of the mechanisms that have been proposed to explain the hypermutation of antibody V regions invoke local error-prone DNA replication or repair (Bertocci et al., 1998; Brenner and Milstein, 1966; Gearhart, 1982; Manser, 1990; Rogerson et al., 1991; Wilson et al., 1998). An alternative theory to those involving error-prone DNA repair has been proposed by Steele and Pollard (1987). They suggested that error-prone reverse transcription of hnRNA creates a cDNA copy of the nascent RNA. Recombination or gene conversion of the resulting mutated cDNAs then replaces the endogenous V gene. This RT model is attractive because it provides a reasonable explanation for many of the mysteries inherent in the somatic mutation process (Steele and Blanden, 2001). The high mutation rate of 10 − 4 to 10 − 3 per base pair per generation (Berek and Milstein, 1987; Kocks and Rajewsky, 1989) is explained by the intrinsic error rates of reverse transcriptases (RTs) that can be as high as 10 − 2 mutations per nucleotide per cycle (Pathak and Temin,

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1990). Somatic mutation tends to occur preferentially in defined sequence motifs called hotspots (Betz et al., 1993a,b; Neuberger and Milstein, 1995; Rogozin and Kolchanov, 1992; Smith et al., 1996). RTs have been shown to have hotspots of mutations with HIV RT displaying a preference for G to A substitutions and sequence context has been shown to affect base substitution fidelity up to 100-fold (Bebenek et al., 1989; Pathak and Temin, 1990; Roberts et al., 1988). While there has been considerable debate about whether there is strand bias, recent studies (Dorner et al., 1998; Milstein et al., 1998) suggest a strand bias at least for those mutations that occur outside of hotspot motifs. Since the RT model depends on the transcript acting as a template for a cDNA, a clear strand bias would also be evident. Thus, the hallmarks of somatic V region hypermutation — the need for transcription, V region localization, the preference for particular hotspots, transitions over transversions, and perhaps strand bias — are explainable through this RT mechanism (Blanden et al., 1998a). The many studies of telomerases (Greider and Blackburn, 1985) and reports of endogenous RTs within human cells (Klenerman et al., 1997) suggest that endogenous RTs exist and are functioning in a similar manner to retroviral RTs. One strategy for testing the RT theory would be to use known inhibitors of retroviral and endogenous RTs. This led us to select 3%-azido3%-deoxythymidine (AZT) and 2%,3%-dideoxycytidine (ddC) for these experiments. Both are 2%,3%-dideoxynucleoside analogs (ddNs) that have been used in treatment of human immunodeficiency virus (HIV) infection (Devineni and Gallo, 1995; Furman et al., 1986; Mitsuya et al., 1985). We examined the effects of AZT and ddC in vivo by assaying antibody affinity maturation and by sequencing endogenous V regions to examine the frequency and characteristics of the mutations in mice treated with these drugs. Since AZT and ddC are prodrugs that must be phosphorylated by cellular kinases to become active, we showed that they were activated in cultured B cells. Neither drug had a notable effect on either the frequency or characteristics of V region mutation, suggesting that RTs are not responsible for V region hypermutation in the mouse.

pended in 2 ml RPMI media with 10% fetal calf serum (Harlan), 4 mM L-glutamine (Sigma), 100 IU/ml penicillin & streptomycin (Mediatech) and 50 mM 2-ME (GibcoBRL). Splenocytes were cultured for 24 h in lipopolysaccharide (LPS) (50 mg/ml, Sigma) and in antiretroviral drugs at 25, 100, or 200 mM concentrations. The cells were then infected with the GFP retrovirus and GFP expression was assayed 48 h later using FACS. Cells were stained with Cy-Chrome anti-mouse CD45R/B220 (RA3-6B2, PharMingen) and approximately 5× 106 cells were assayed per condition. The NSO LC1 cell line stably transfected with a mouse g2a heavy chain construct has been described previously (Zhu et al., 1996, 1995). These cells were pretreated for 3 h with RT-inhibitors, AZT or ddC, and then infected with virus in the presence of AZT and ddC. Approximately 40,000 cells were assayed per condition.

2.2. Drug treatment and serum analysis of mice Two experiments were carried out. In the first experiment, nine mice were examined. In the second experiment for which the data is shown, 15 C57BL/6 6- to 8-week-old female mice (Jackson Labs) were used. Drugs were administered as previously described (Pettoello-Mantovani et al., 1998). Treatment began 3 days (day −3) before the primary immunization. The five AZT mice consumed, on average, 3.7 g feed/day (versus 3.9 g for control mice), which is around 147 mg/kg/day AZT. The five ddC mice consumed an average of 3.8 g feed/day, which is around 61 mg/kg/day ddC. AZT (Glaxo Wellcome) and ddC (Roche) for the mouse studies were obtained from the Weiler Pharmacy at the Einstein Hospital. Mice were bled from the tail on day 0, injected intraperitoneally with 100mg alum-precipitated sterile nitrophenylated (NP) chicken gamma globulin (CGG) (NP22-CGG; BioSearch Technologies, Inc.) in 100 ml saline and then bled of 100–200 ml blood from the tail vein on days 10, 20, 31 and 45. The sera were assayed by enzyme-linked immuosorbent assay as previously described (Wiesendanger et al., 2000). Highaffinity anti-NP antibodies were measured on NP2.5-bovine serum albumen coated plates (BioSearch Technologies, Inc.) using anti-isotype antisera (Pharmingen).

2. Materials and methods

2.3. Sequencing of the V region genes 2.1. Prodrug acti6ation and RT inhibition The green fluorescent protein (GFP) expressing pLEIN Retroviral Vector (Clontech) packaged in the PT67 cell line (Clontech) was used to show that AZT (Sigma) and ddC (Sigma) were phosphorylated in B cells. Spleen cells were depleted of red blood cells by lysis using 0.17 M NH4Cl (pH 7.4). Cells were resus-

The mice were boosted with hapenated chicken gamma globulin 39 days after the primary immunization. Six days later, two mice each from the control, AZT, and ddC groups were sacrificed and their spleens were harvested. cDNA was synthesized using oligo(dT) primed SuperScript reverse transcription (GibcoBRL). Nested polymerase chain reaction (PCR) was per-

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formed with high-fidelity Pfu DNA polymerase (Stratagene) to amplify VH186.2 joined to the immunoglobulin (Ig)G1 constant region as previously described (Matsumoto et al., 1996). Both rounds of PCR were performed for 30 cycles of 1 min at 94 °C, 2 min at 55 °C, and 3 min at 72 °C. For the first round, the 5% VH186.2 primer was CAT GCT CTT CTT GGC AGC AAC AGC, and the Cg1 primer was GTG CAC ACC GCT GGA CAG GGA TCC. The second-round VH186.2 primer was the 5% region of the V gene itself (CAG GTC CAA CTG CAG CAG), while the second-round Cg1 primer was AGT TTG GGC AGC AGA. Amplified DNA was gel purified on a 1.2% agarose gel with Qiaex II (Qiagen) and cloned into the plasmid vector, pCR®4Blunt-TOPO (Invitrogen) before transformation into TOP10 bacteria. Plasmid DNA was prepared using the QIAprep miniprep kit (Qiagen). Both DNA strands were sequenced with T3 and T7 primers using an ABI automatic sequencer model 377. The sequences were then analyzed and compared with the various members of the V186.2 and V3 Family using MacDNASIS (Hitachi Software). Genbank Accession numbers: AF312421– AF312488, AF312668.

2.4. Statistical analysis The mutation frequency between AZT-treated (P= 0.056), ddC-treated (P =0.069), and control groups are not significantly different as measured by analysis of variance and the post-hoc Dunnett t-test (two-sided). The statistical analysis was performed in SPSS for Windows, Release 9.0.

Table 1 GFP expression assayed by FACS Sample

Live cells

NSO NSO NSO NSO NSO

4.2×104 4.2×104 4.1×104 4.2×104 4.3×104

16.68 0.45 0.03 11.42 3.41

0 97 \99.5 32 80

1.6×105 4.5×105 3.7×105 4.1×105 8.3×105 7.9×105 4.6×105

1.75 B0.01 B0.01 B0.01 1.01 0.32 0.06

0 \99.5 \99.5 \99.5 31 78 96

control AZT 25 AZT 200 ddC 25 ddC 200

Spleen Spleen Spleen Spleen Spleen Spleen Spleen

control AZT 25 AZT 100 AZT 200 ddC 25 ddC 100 ddC 200

% GFP-positive % Inhibition

NSO cells are gated for live cells and primary splenocytes are gated for blasted cells that are B220+ (Cy-Chrome). GFP integration and expression is dependent on RT activity. RT inhibitors AZT and ddC demonstrate a dose-dependent inhibition of GFP expression, demonstrating that the prodrug is converted to an active form that inhibits RT in B cells.

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3. Results

3.1. Acti6ity of AZT and ddC in B cells AZT and ddC are among the most potent RT inhibitors of the ddNs in replicating cells (Gao et al., 1993a, 1994b; Shirasaka et al., 1995). Since both are prodrugs, we determined whether they were activated in B cells. To do this, we first examined drug toxicity on mouse g2a-transfected NSO cells, a mouse myeloma cell line, which we have previously shown can carry out V region mutation in vitro (Green et al., 1998). Killing curves revealed that 160 mM ddC killed 10% of the NSO mouse myeloma cells, whereas AZT killed 5% of the cells at 300 mM (data not shown). While the finding of toxicity suggests that these prodrugs are being processed in this tumor cell line, we confirmed this by assaying a retroviral test plasmid that contains the GFP gene. This vector is derived from the Moloney murine leukemia virus (MoMLV) and relies on reverse transcription of the viral genome in order to integrate itself into the cell genome so that the GFP can be expressed. FACS analysis demonstrates that GFP expression occurs in approximately 17% of g2a-transfected NSO cells (Table 1). The drugs inhibited GFP expression in a dose-dependent fashion with 25 mM AZT decreasing GFP expression by 97% and 200 mM AZT decreasing GFP expression by \ 99.5%. ddC-treated cells also showed dose-dependent decreases with 25 mM decreasing GFP expression 32% and 200 mM decreasing GFP expression 78%. The different efficacy of the drugs in this assay presumably reflects the relative resistance of the MoMLV RT to ddC (Goulaouic et al., 1994). There is no straightforward way to assay the ability of B cells to process the prodrugs in vivo. However, we used the same retroviral GFP expression assay to examine primary B cells in vitro. Freshly harvested splenic B cells were cultured with LPS for 24 h in the presence of various doses of AZT and ddC, and then infected and assayed for GFP expression 48 h later. The primary B220+ B cells were less susceptible to infection than the NSO cell line and GFP expression was observed in only 1.75% of the cells. Pretreatment with AZT at 25, 100 and 200 mM were all extremely effective and decreased expression to less than 0.01% of cells (Table 1). The effects of ddC were more dose dependent with 25 mM decreasing expression 31%, 100 mM decreasing GFP expression by  80% and 200 mM decreasing expression 96%. Similar results were also obtained for B220+ PNAhi cell populations, except that smaller numbers of cells were assayed because of the difficulty of obtaining large numbers of germinal center B cells and keeping them alive in vitro (data not shown).

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Fig. 1. Serum titers from 15 mice were measured immediately prior to NP immunization (1°) and during the course of the primary response (days 10, 20, 31, and 39). The mice were boosted on day 39 (2°) and their serum titer was assayed on day 45 prior to sacrifice. Titers were determined on NP2.5 plates using twice background as the cutoff. The data at each time point is the average of five mice in each group: AZT mice (), ddC mice ( ), and Control mice ( ).

3.2. Affinity maturation We examined the effect of these two RT inhibitors on somatic V region mutation in vivo, first by studying the affinity maturation of the serum antibody responses and then by sequencing heavy chain V regions from drug-treated and untreated-control mice. We have confirmed the findings of many others (Smith et al., 1997; Takahashi et al., 1998; Weiss and Rajewsky, 1990) that C57BL/6 mice respond to immunization with alum precipitated NP coupled to chicken gamma globulin with IgM and then IgG responses that increase during the primary response (Wiesendanger et al., 2000). Groups of five mice were examined. The AZT and ddC mice had similar primary and secondary IgM responses to NP as the untreated mice (data not shown). As has been reported by others, the highaffinity IgG levels increased during the course of observation even as the total anti-NP antibodies decreased late in the primary response. The levels and increase in high-affinity IgG were indistinguishable in the AZT, ddC, and control mice (Fig. 1). Similar results were obtained in a preliminary experiment using groups of three mice (data not shown).

3.3. Mouse sequence data The lack of effect of either drug on the affinity maturation of the immune response suggests that they are not affecting somatic mutation in vivo. To confirm that AZT and ddC were not having an effect on the frequency or characteristics of the base changes that occur as a result of V region mutation, heavy chain V regions of antibodies involved in the anti-NP response

were sequenced. Members of the J558 V region family dominate the anti-NP response in C57BL/6 mice (Gu et al., 1991; Weiss and Rajewsky, 1990). The V186.2 subfamily was chosen for PCR amplification and sequence analysis because it is well established that the endogenous response to the NP hapten is dominated by this subfamily (Gu et al., 1991; Weiss and Rajewsky, 1990). Furthermore, affinity maturation of V186.2 primarily depends on a Trp to Leu mutation at position 33 in the first complementarity determining region (Rajewsky et al., 1987). Since mutations at residue 33 are positively selected because they interact with antigen, codon 31 is of particular interest because it contains a G hotspot on the coding strand and, at the next base, a G hotspot on the non-coding strand, and both Gs are embedded in RG6 YW motifs. Mutations at this codon do not increase the affinity of NP binding (Allen et al., 1988) and should not be selected for by antigen. Therefore, mutations at residue 31, and at other locations in the framework regions that are not likely to be selected for by antigen, should reveal quantitative or qualitative changes in the mutational process. The control, AZT-treated and ddC-treated mice demonstrate a dominant V186.2 response. Looking at the secondary response sequence data (Table 2) of the control, AZT, and ddC mice, the percent of mutated V regions are virtually the same. Although the frequency of mutation in the control is slightly higher than in the AZT- and ddC-treated mice, this is not statistically significant and furthermore the characteristics of the mutational process are not different. As expected from the serological results (Fig. 1), codon 33 accumulates a large number of mutations (Fig. 2) with the affinity enhancing G“T replacement mutation being the most prevalent (Fig. 2 shows the frequency of all mutations at each base and Table 2 presents the frequency for the whole codon). This G “ T transversion represents 8% of the total mutations examined in the control mice, and 9 and 13% of those occurring in AZT and ddC sequences, confirming an active anti-NP response that undergoes affinity maturation in both AZT- and ddCtreated mice. In all groups, the R/S ratios in the CDRs are at least three times those in the framework regions (data not shown), also indicating that these V regions were positively selected. The hotspot mutations at codon 31 are also occurring at a high frequency (Fig. 2) with 8–10% of the mutations arising at this codon (Table 2). The preference for the hotspot RGYW motifs throughout the V region is readily apparent in all three groups in that mutations in the G of the RGYW motif account for between 26 and 27% of all mutations in each group. As with the responses in the control mice, the most commonly mutated base in AZT- and ddC-treated mice is G (44%, 46 and 56%, respectively) with T being the least mutated (3%, 11 and 7%, respectively). Similar

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sequence data were obtained in an independent experiment with B220+ PNAhi sorted splenocytes from nine mice (data not shown).

4. Discussion Although many mechanisms have been proposed to explain somatic V hypermutation, none of them fully account for the many unusual characteristics of this process. Recent studies have revealed that mismatch repair plays a role in V region mutation (Reynaud et al., 1999; Wiesendanger et al., 2000) and the AID RNA editing enzyme is also involved (Muramatsu et al., 2000; Revy et al., 2000). Nevertheless, it has been difficult to explain the very high rates of single base changes, the characteristics of the hotspots and the apparent restriction of the mutational target to a region starting just upstream of the transcriptional start site and ending  1.5 kb further downstream. The proposal by Steele and his colleagues that an endogenous reverse transcriptase could be responsible for the high rate of mutation of antibody V region genes (Blanden et al., 1998b; Steele and Blanden, 2001) does provide an explanation for many of the characteristics of V region mutation. Since gene conversion-like events are responsible for creating the large repertoire of V region sequences in chickens and rabbits (Reynaud et al., 1996; Winstead et al., 1999), and gene conversion could be responsible for replacing the endogenous V region with the error containing copy in the RT model, this theory also pulls together the generation of diversity in many different species (Blanden et al., 1998a). The implications of this theory are also consequential to HIV-positive patients undergoing anti-retroviral therapy. Nevertheless, there is no direct experimental evidence

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for (or against) the RT model, nor has the putative RT been identified in germinal center B cells. To begin to test the RT model, we have examined whether known inhibitors of RTs inhibit V region hypermutation in vivo. The GFP results in the primary splenocytes combined with the results from the cultured cell line led us to conclude that AZT and ddC are active in B cells in vivo and in vitro. Our in vivo studies employed doses of approximately 147 and 61 mg/kg/ day of AZT and ddC, respectively. Depending on the methodology used, previous studies have shown in vivo inhibition of HIV with 50% effective doses (ED50) between 2 and 20 mg/kg/day for AZT (Rabin et al., 1996), with ddC’s ED50 being about 0.5–0.7 mg/kg (Sato et al., 1995). The doses used in this study were well above those required in mice to inhibit HIV replication. Assuming that the drugs are fully absorbed and evenly distributed throughout the mice, these doses achieve a target concentration of 550 mM AZT/day and 147 mM ddC/day. As with the studies described here, most studies use higher doses to ensure efficacy, and have shown that at an AZT dose of 160–200 mg/kg/ day there is no overt toxicity to mice (McCune et al., 1990). ddC has been used at 80 mg/kg/day with higher concentrations in feed not possible due to aversion by the mice (Basham et al., 1991). Even at these very high doses, there was affinity maturation of the antibody response to the very well studied hapten NP, suggesting that the antibody response was not affected by the drugs. In addition, the frequency and characteristics of V region mutations, even in codons where mutations are unlikely to be selected for by antigen, are the same in animals that were exposed to the AZT and ddC, and in the control mice. Since this sort of analysis has revealed quantitative differences in the frequency of mutations in AID-deficient mice and patients (Muramatsu et al., 2000; Revy et al., 2000) and qualitative

Table 2 Analysis of sequences

Number of sequences analyzed % Mutated sequences Total mutations Mutation frequency % Mutations in hotspots % Mutations in comp-hotspots % Mutations in codon 31 % Mutations in codon 33 G “T Ts/Tv ratio R/S ratio total

Control mice

AZT-treated

ddC-treated

28 93 187 2.45×10−2 27 9 8 8 1.2 5.0

19 89 81 1.56×10−2 26 6 10 9 1.1 3.1

22 95 98 1.63×10−2 27 8 10 13 1.8 2.3

Mutations that appear more than once in a genealogy are only counted once. Sequences were placed in a genealogy based on finding an identical CDR3 region and shared mutations. Hotspots are mutations only in the G of RG6 YW and the C of WRC6 Y. Only the 276 bp between the 5% primer and the CDR3 region were analyzed. When doublets/triplets occurred, each mutation was analyzed independently of the others with respect to R/S ratio. Ratios of Ts/Tv and R/S are calculated after excluding the affinity enhancing codon 33 G to T mutation, which is a result of focused affinity selection and would skew the Ts/Tv results (Ts/Tv would be 1.0, 0.9, and 1.3, respectively). These data are the average of two independent mice in each group. The mutation frequency between the three groups are not significantly different (P\0.05, see Section 2).

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Fig. 2. Schematic of the distribution of mutations along the 276 bp between the 5% primer and the CDR3 region of v186.2. A large number of mutations occur in hotspot codon 31 (AGC), which contains both an RGYW and a WRCY motif. Most mutations occur in the G (labeled 31). The affinity enhancing codon 33 in CDR1 is also labeled. Each bar represents the percentage of mutations that occur at that position in each of the three groups of mice (control, AZT-treated, and ddC-treated). The CDR1 and CDR2 region are depicted underneath each graph. These data are the average of two independent mice in each group with 187 mutations in the control, 81 in the AZT-treated, and 98 in the ddC-treated. These data exclude genealogies.

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differences in the characteristics of the base changes in mice deficient in mismatch repair enzymes (Reynaud et al., 1999; Wiesendanger et al., 2000), we would have expected to find differences between the drug-treated and control mice if a drug-sensitive RT were playing a crucial role in V region mutation. A majority of RT-inhibitor studies have been conducted on HIV-RT since drug action, interaction, and resistance are important issues in the treatment of AIDS. The observed 50% inhibitory concentration (IC50) of HIV-RT by AZT is 0.06 mM. In resistant strains, the IC50 has been shown to increase to 0.45–2 mM (Gao et al., 1993b; Richman et al., 1994). The IC50 for ddC is 0.07– 0.5 mM and drug resistance increases it only 10- to 15-fold (Gao et al., 1994a). In addition, RTs that become resistant to one of these drugs generally do not become cross-resistant to the other; thus, the use of both drugs provided us with wider coverage of putative endogenous RTs (Foli et al., 1996; Gao et al., 1993a; Richman et al., 1994; Ueno and Mitsuya, 1997). Furthermore, multiple examples of resistance to either AZT or ddC caused increased sensitivity to the other drug. Of prime importance to the RT theory of V region mutation is the presence of endogenous RTs. Recent work by Klenerman et al. demonstrates the ability of mammalian cells to use an unknown endogenous reverse transcriptase to convert a RNA virus (LCMV which lacks RT) into cDNA, which is then integrated into the genome (Klenerman et al., 1997). They found that AZT inhibited integration in a dose-dependent manner, thus demonstrating the inhibition of an unknown endogenous RT with this class of inhibitor. Experiments on the Telomerase RT and attempts to inhibit its function through drug inhibition provide further precedence for these experiments (Strahl and Blackburn, 1994, 1996). These studies, and those already discussed showing that AZT and ddC have somewhat different and complementary abilities to inhibit many different viral RTs, make it more likely that these drugs would inhibit a cellular-RT-involved in V region mutation, if it existed. Another interesting aspect of these inhibitors is their effect on cellular polymerases. DNA polymerase b and DNA polymerase g have been shown to promote the incorporation of activated AZT and ddC, thereby inhibiting enzyme activity (Cherrington et al., 1994; Starnes and Cheng, 1987). Thus, enzymes involved in DNA repair (DNA polymerase b) and mitochondrial DNA synthesis (DNA polymerase g) are also assayed in the experiments reported in this paper. AZT has a Ki for polymerase b of 140 mM and for polymerase g of 18.3 mM. The Ki for ddC is 1.32– 2.6 mM for polymerase b and 0.016– 0.034 mM for polymerase g. Since effects were not detected, this suggests that neither polymerase b nor polymerase g is involved in the mechanism of somatic mutation in the cultured cells. These data are consistent with the recent polymerase b knock-out ex-

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periments (Esposito et al., 2000) and may provide insight into some of the many novel polymerases now being investigated (Poltoratsky et al., 2000) We conclude that it is very unlikely that a reverse transcriptase is required in V region mutation, although we cannot exclude the possibility that a RT that is completely different than those known to exist and is not subject to inhibition by nucleoside analogues, is playing a role in V region hypermutation.

Acknowledgements The authors would like to thank Dr Margrit Wiesendanger, Dr Jeffrey Pollard, Dr Vinayaka Prasad, and Dr Harris Goldstein for helpful suggestions. This work was supported by grants from the National institutes of Health: T32 GM 07288 (S.Z.S.), 5T32 CA 09173 (P.D.B.), AI 43937 (M.D.S.). Dr Scharff is also supported by the Harry Eagle Chair provided by the Women’s Division of the Albert Einstein College of Medicine. References Allen, D., Simon, T., Stablizky, F., Rajewsky, K., Cumano, A., 1988. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 7, 1995 – 2001. Basham, T., Holdener, T., Merigan, T., 1991. Intermittent, alternating, and concurrent regimens of zidovudine and 2%-3% dideoxycytidine in the LP-BM5 murine induced immunodeficiency model. J. Infect. Dis. 163, 869 – 872. Bebenek, K., Abbotts, J., Roberts, J.D., Wilson, S.H., Kunkel, T.A., 1989. Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase. J. Biol. Chem. 264, 16948 – 16956. Berek, C., Milstein, C., 1987. Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96, 23 – 41. Bertocci, B., Quint, L., Delbos, F., Garcia, C., Reynaud, C.-A., Weill, J.-C., 1998. Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process. Immunity 9, 257 – 265. Betz, A.G., Neuberger, M.S., Milstein, C., 1993a. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14, 405 – 411. Betz, A.G., Rada, C., Pannell, R., Milstein, C., Neuberger, M.S., 1993b. Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots. Proc. Natl. Acad. Sci. USA 90, 2385 – 2388. Blanden, R.V., Rothenfluh, H.S., Zylstra, P., Weiller, G.F., Steele, E.J., 1998a. The signature of somatic hypermutation appears to be written into the germline IgV segment repertoire. Immunol. Rev. 162, 117 – 132. Blanden, R.V., Rothenfluth, H.S., Steele, E.J., 1998b. On the possible role of natural reverse genetics in the V gene loci. Curr. Top. Microbiol. Immunol. 229, 21 – 32. Brenner, S., Milstein, C., 1966. Origin of antibody variation. Nature 211, 242 – 243. Cherrington, J.M., Allen, S.J., McKee, B.H., Chen, M.S., 1994. Kinetic analysis of the interaction between the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, ddCTP,

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