Cellular inhibitors of long interspersed element 1 and Alu retrotransposition.
ABSTRACT Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent approximately 17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further approximately 11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.
- [show abstract] [hide abstract]
ABSTRACT: APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) is an innate intracellular antiretroviral factor that can inhibit viral retroelements such as retroviruses and hepadnaviruses. However, it is unknown whether it can act on non-viral substrates. Retrotransposons are transposable elements that cumulatively account for about one third of the human genome. They are commonly classified in long terminal repeat (LTR) retrotransposons, which are strongly homologous to retroviruses, and non-LTR retrotransposons also known as L1 elements or LINE-1 (long interspersed nucleotide element-1) elements. Most of the L1 elements are defective and only a small number are very active in vivo, but they are responsible for nearby all of the retrotransposition in the human population. The cloning of active human L1 elements has allowed the development of tissue culture-based assays for measuring their retrotransposition potential. We used such an assay to demonstrate that APOBEC3G, which impairs the replication of exogenous retroelements, does not affect the replication of endogenous L1 retrotransposons.Journal of Biological Chemistry 11/2004; 279(42):43371-3. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.Current Biology 04/2006; 16(5):480-5. · 9.49 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The cytidine (C) to uridine (U) editing of apolipoprotein (apo) B mRNA is mediated by tissue-specific, RNA-binding cytidine deaminase APOBEC1. APOBEC1 is structurally homologous to Escherichia coli cytidine deaminase (ECCDA), but has evolved specific features required for RNA substrate binding and editing. A signature sequence for APOBEC1 has been used to identify other members of this family. One of these genes, designated APOBEC2, is found on chromosome 6. Another gene corresponds to the activation-induced deaminase (AID) gene, which is located adjacent to APOBEC1 on chromosome 12. Seven additional genes, or pseudogenes (designated APOBEC3A to 3G), are arrayed in tandem on chromosome 22. Not present in rodents, this locus is apparently an anthropoid-specific expansion of the APOBEC family. The conclusion that these new genes encode orphan C to U RNA-editing enzymes of the APOBEC family comes from similarity in amino acid sequence with APOBEC1, conserved intron/exon organization, tissue-specific expression, homodimerization, and zinc and RNA binding similar to APOBEC1. Tissue-specific expression of these genes in a variety of cell lines, along with other evidence, suggests a role for these enzymes in growth or cell cycle control.Genomics 04/2002; 79(3):285-96. · 3.01 Impact Factor
Cellular inhibitors of long interspersed element 1
and Alu retrotransposition
Hal P. Bogerd†, Heather L. Wiegand†, Amy E. Hulme‡, Jose ´ L. Garcia-Perez‡, K. Sue O’Shea§, John V. Moran‡,
and Bryan R. Cullen†¶
†Center for Virology and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710; and Departments
of‡Human Genetics and Internal Medicine and§Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109
Communicated by Wolfgang K. Joklik, Duke University Medical Center, Durham, NC, April 24, 2006 (received for review February 27, 2006)
Long interspersed element (LINE) 1 retrotransposons are major
LINE-1 ORF2 protein is also responsible for the mobility of Alu
elements, which constitute a further ?11% of genomic DNA.
Representative members of each element class remain mobile, and
deleterious retrotransposition events can induce spontaneous ge-
netic diseases. Here, we demonstrate that APOBEC3A and
APOBEC3B, two members of the APOBEC3 family of human innate
antiretroviral resistance factors, can enter the nucleus, where
LINE-1 and Alu reverse transcription occurs, and specifically inhibit
both LINE-1 and Alu retrotransposition. These data suggest that
the APOBEC3 protein family may have evolved, at least in part, to
defend the integrity of the human genome against endogenous
APOBEC3 protein ? mutagenesis ? retrotransposon ? genome stability ?
and retrotransposons lacking LTRs (non-LTR retrotrans-
posons). Non-LTR retrotransposons can be subdivided further
into long interspersed elements (LINEs) and short interspersed
elements (SINEs). LINE-1, the most common human LINE, is
transcribed by RNA polymerase II to give an ?6-kb mRNA that
encodes two proteins, ORF1p (or p40) and ORF2p, which are
required for retrotransposition (1, 2). In contrast, the most
common human SINE, Alu, is transcribed by RNA polymerase
III to give an ?300-nt noncoding RNA (3). Alu retrotranspo-
sition depends on the LINE-1 ORF2p (4).
Humans are subject to infection by a limited number of
exogenous retroviruses and contain few, if any, biologically
active LTR retrotransposons, although a small number of struc-
turally similar endogenous retroviruses may remain viable (5, 6).
However, humans contain ?100 intact LINE-1 elements (7, 8),
and de novo LINE-1 and short interspersed element retrotrans-
position events occur in ?10% of human genomes per genera-
tion (9, 10). Although they are frequently innocuous, these
retrotransposition events account for ?0.2% of all spontaneous
deleterious human mutations (11, 12). Moreover, LINE-1 and
Alu have accumulated to very high levels in the human genome.
LINE-1 elements now constitute ?17% of the human genome,
and the ?106copies of Alu constitute a further ?11% (5).
The ability of human APOBEC3G (A3G) to function as an
innate inhibitor of exogenous retroviruses was first noted during
studies analyzing the HIV type 1 (HIV-1) Vif protein (6, 13).
These experiments revealed that A3G is a potent inhibitor of
Vif-deficient, but not wild-type, HIV-1 replication. In the ab-
sence of Vif, A3G is specifically packaged into progeny virion
particles and then interferes with reverse transcription during
subsequent infections (6, 13). Although the mechanisms under-
lying this inhibition are not fully defined, A3G is a cytidine
deaminase (CDA) that edits dC residues to dU on nascent DNA
minus strands during reverse transcription (14–16). This activity
etroelements can be divided into at least three classes:
exogenous retroviruses, retrotransposons containing LTRs,
induces extensive mutagenesis of the HIV-1 provirus and may
destabilize incomplete reverse transcripts.
The human APOBEC3 protein family consists of at least five
active members that contain one or two consensus CDA active
sites (6, 17). Two sites are found in APOBEC3B (A3B, 382 aa),
APOBEC3F (A3F, 373 aa), and A3G (384 aa), and one is found
in the smaller APOBEC3A (A3A, 199 aa) and APOBEC3C
(A3C, 190 aa) proteins. Although A3B, A3F, and A3G can all
inhibit Vif-deficient HIV-1 replication, A3A is not active against
HIV-1; A3C is only weakly active but does inhibit Vif-deficient
simian immunodeficiency virus (6, 18, 19).
The human APOBEC3 proteins are undergoing rapid adap-
tive evolution, implying that these gene products are in an
evolutionary race with some form of deleterious retroelement(s)
(20, 21). Human APOBEC3 proteins can inhibit exogenous
retroviruses of non-human origin as well as several LTR retro-
transposons, thus suggesting that these retroelements could be a
source of selective pressure (6, 22–24). Other potential drivers of
this adaptive evolution include the human non-LTR retrotrans-
posons and, in particular, LINE-1 and Alu. Here, we demon-
strate that two members of the human APOBEC3 family, A3A
and A3B, can indeed inhibit both LINE-1 and Alu mobility.
Subcellular Localization of APOBEC3 Proteins. Although human
A3G can inhibit several exogenous retroviruses and LTR ret-
rotransposons (6), it has no effect on LINE-1 mobility (24, 25).
Unlike retroviruses and LTR retrotransposons, which undergo
cytoplasmic reverse transcription, LINE-1 RNA is reverse-
transcribed in the nucleus (26, 27), and A3G has previously been
reported to be restricted to the cytoplasm (14). If the inability of
A3G to inhibit LINE-1 retrotransposition reflects this compart-
mentalization, then APOBEC3 proteins that enter the nucleus
might be more effective inhibitors of LINE-1 retrotransposition.
Because the exclusion size for passive diffusion through the
nuclear pore complex is ?40 kDa (28), we asked whether A3A
and A3C, which fall below this limit, would enter the nucleus. As
shown in Fig. 1A, although A3G and A3F are excluded from
nuclei, A3A and A3C are indeed found in both the nucleus and
cytoplasm. This result is consistent with the hypothesis that A3A
and A3C can enter nuclei by passive diffusion. Unexpectedly,
A3B appeared nuclear at steady state. The 382-aa A3B protein
is too large to diffuse into the nucleus (28), implying that A3B
contains a nuclear localization signal (NLS).
As a first step toward mapping the A3B NLS, we divided A3B
into two nonoverlapping segments, extending from amino acid 1
to 192 and from amino acid 193 (a methionine) to 382, that
resemble single CDA domain APOBEC3 proteins. As shown in
Conflict of interest statement: No conflicts declared.
HIV-1, HIV type 1; NLS, nuclear localization signal; HA, hemagglutinin; IAP, intracisternal A
¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
June 6, 2006 ?
vol. 103 ?
no. 23 www.pnas.org?cgi?doi?10.1073?pnas.0603313103
Fig. 1A, the N-terminal half of A3B, termed N-A3B, localized to
the nuclei of expressing cells, whereas the C-terminal half,
termed C-A3B, gave the same diffuse subcellular localization
seen with A3A and A3C. Therefore, we conclude that the A3B
NLS is located in the N-terminal 192-aa segment. The analogous
N-terminal (N-A3G, 196 aa) and C-terminal (C-A3G, 188 aa)
segments of A3G both showed a diffuse subcellular localization
(Fig. 1A). This result is consistent with the hypothesis that A3G
lacks a NLS and suggests that the cytoplasmic localization of
full-length A3G (Fig. 1A) indeed results from the inability of the
?42-kDa A3G protein to enter nuclei by passive diffusion.
At first glance, the nuclear localization of A3B appears
inconsistent with reports that demonstrate that A3B, like A3G,
can assemble into the virions of several exogenous retroviruses
in the cytoplasm and then inhibit their replication (19, 29, 30).
To test whether A3B is actually a nucleocytoplasmic shuttle
protein, we fused COS7 cells expressing hemagglutinin (HA)
epitope-tagged A3B to HeLa-GSN cells that express a fusion
protein consisting of GFP linked to the SV40 T antigen NLS
(31). As shown in Fig. 1B, the HA-tagged A3B protein moved
from COS7 nuclei to HeLa-GSN nuclei that were present in the
resultant heterokaryons. In contrast, the GFPT-NLS fusion
protein, which lacks a nuclear export signal (NES), failed to
move from HeLa-GSN nuclei to COS7 nuclei. We therefore
conclude that A3B is a nucleocytoplasmic shuttle protein that
contains a NLS and a NES.
A3A and A3B Inhibit LINE-1 Retrotransposition. The ability of A3A,
A3B, and A3C to enter nuclei suggested that one or more of
these proteins might be able to interfere with the retrotranspo-
sition of human LINE-1. To explore this hypothesis, we tested
whether an engineered LINE-1 element harboring a retrotrans-
position indicator (pJM101?L1.3) (32, 33) was affected by
APOBEC3 protein expression. The retrotransposition indicator
cassette consists of a neo gene in the antisense orientation
(relative to LINE-1) that is disrupted by an intron in the sense
orientation (32, 34). Therefore, neo expression requires LINE-1
transcription, removal of the intron by splicing, reverse tran-
scription, and integration followed by expression of the now
intact neo gene. Cotransfection of HeLa cells with pJM101?L1.3
and a plasmid encoding an APOBEC3 protein revealed that
A3A and A3B are effective inhibitors of LINE-1 retrotranspo-
sition (Fig. 2). The A3C protein exerted a modest but significant
inhibitory effect on LINE-1 mobility, whereas A3G and A3F had
little effect on retrotransposition. The observed inhibition was
not due to nonspecific toxicity, because we have previously
shown that the APOBEC3 proteins do not reduce the number of
G418-resistant colonies obtained after cotransfection into HeLa
cells with a neo expression plasmid (23). Comparison of the
effect of APOBEC3 proteins on LINE-1 retrotransposition with
is essentially no correlation. All APOBEC3 proteins were ex-
pressed at comparable levels (Fig. 6, which is published as
supporting information on the PNAS web site).
Inhibition of LINE-1 Retrotransposition Occurs in the Absence of
Hypermutation. Inhibition of retrovirus or LTR retrotransposon
replication by wild-type A3G is associated with hypermutation
of any resultant integrated proviruses (6, 14–16, 24). In contrast,
inhibition of hepatitis B virus replication by A3G seems to occur
in the absence of dC deamination (36, 37), and mutants of A3G
that are unable to edit retain substantial inhibitory activity
against HIV-1?Vif (38). Extensive sequencing of LINE-1 ele-
ments that retrotransposed in the presence of A3A, A3B, A3C,
or A3G failed to detect G-to-A hypermutation (Fig. 7, which is
published as supporting information on the PNAS web site).
Therefore, inhibition of LINE-1 retrotransposition by A3A and
A3B does not involve editing, or edited nuclear reverse tran-
scripts are rapidly degraded.
Although A3G, like A3B and A3F, contains two consensus
CDA active sites, only the C-terminal site is enzymatically active;
the N-terminal site appears to be involved in mediating selective
incorporation of A3G into virion particles (38–40). To deter-
mine whether an enzymatically active CDA consensus site is
required for inhibition of LINE-1 retrotransposition by A3A or
A3B, we used mutagenesis to inactivate the CDA active site that
is present in A3A or one of the two CDA active sites that are
present in A3B. The introduced mutations, which changed the
glutamic acid residue in the CDA consensus active site (His-X-
Glu-X25–28-Pro-Cys-X2–4-Cys) (6) to glutamine, have previously
were transfected with plasmids expressing the indicated wild-type or mutant
APOBEC3 protein bearing a C-terminal HA epitope tag. At 48 h after trans-
fection, cells were fixed and incubated with a mouse monoclonal anti-HA
antibody, followed by tetramethylrhodamine B isothiocyanate-conjugated
goat anti-mouse antiserum, and visualized by fluorescence. (B) Nucleocyto-
plasmic shuttling was visualized by using heterokaryons (45). Briefly, COS7
cells were transfected with a plasmid expressing A3B-HA. Two days later, the
COS7 cells were mixed with HeLa-GSN cells, which express GFP fused to a NLS
(31). The mixed culture was then treated with cycloheximide and fused by
using polyethylene glycol. A3B-HA was detected as described above.
Nuclei were visualized by staining with DAPI; GFP was detected by intrinsic
Subcellular localization of human APOBEC3 proteins. (A) HeLa cells
Bogerd et al.
June 6, 2006 ?
vol. 103 ?
no. 23 ?
been shown to block CDA enzyme activity (38). We also asked
whether the N- or C-terminal halves of A3B (Fig. 1A) could
function as CDAs and?or inhibit LINE-1 retrotransposition.
To address whether the various A3A and A3B mutants have, in
fact, lost the ability to function as CDAs, we used a previously
the ability of an expressed gene product to mutate the RNA
be detected by screening for the frequency of rifampicin-resistant
(rifR) colonies. Expression of A3G or A3F has previously been
shown to enhance the frequency of such mutations (40).
As shown in Fig. 3A, wild-type A3A and A3B both acted as
potent mutators in E. coli. Sequencing the rpoB gene in the
resistant colonies that were obtained in the presence of either
A3A or A3B revealed selective C-to-T mutagenesis with a
consensus target sequence of TC* (data not shown). This
consensus is the same one previously reported for A3B and A3F
but differs from the consensus target sequence for A3G, which
is CC* (19, 29, 40, 41).
Mutation of the single CDA consensus site in A3A (A3Am)
blocked CDA activity as expected. Of interest, mutation of the
N-terminal CDA consensus site in A3B (A3Bm1) had no effect
on editing by A3B, whereas mutation of the C-terminal consen-
sus site (A3Bm2) entirely blocked A3B editing (Fig. 3A). Con-
sistent with the hypothesis that the C-terminal A3B consensus
site is necessary and sufficient for CDA enzyme activity, the
N-terminal 192-aa segment of A3B (N-A3B) did not display any
mutator activity in bacteria, whereas the C-terminal 190-aa
segment (C-A3B) was highly active unless the CDA active site
was mutated (Fig. 3A). Importantly, all of these proteins were
expressed at comparable levels in bacteria (Fig. 8A, which is
published as supporting information on the PNAS web site).
Therefore, we conclude that A3A and A3B both contain a single
enzymatically active CDA consensus site and that, in A3B, this
site is located in the C-terminal half. A3B is therefore similar to
A3G, which also contains only a single enzymatically active CDA
consensus site located in the C-terminal half of the protein
Having constructed several mutants of A3A and A3B that lack
(A3Am, A3Bm2, N-A3B, N-A3Bm1, and C-A3Bm2) or retain
(A3Bm1 and C-A3B) CDA activity, we next asked whether these
proteins would inhibit LINE-1 retrotransposition. As a control,
APOBEC3 proteins. (A) The effect of APOBEC3 proteins on HIV-1?Vif infectiv-
ity was quantified as described in ref. 41. Data are presented relative to a
culture lacking any APOBEC3 protein (positive), which was designated as
100% activity. Data are the average of at least three experiments, with
standard deviation indicated. The relative expression of each APOBEC3 pro-
tein was comparable (Fig. 6). Because the effect of APOBEC3 proteins on the
infectivity of Vif-deficient HIV-1 was measured by using 293T cells, although
their effect on LINE-1 retrotransposition was measured in HeLa cells, these
results are only qualitatively comparable. (B) Representative experiment
showing the relative number of G418-resistant colonies obtained after selec-
tion of HeLa cells transfected with the LINE-1 retrotransposition indicator
construct pJM101?L1.3 or the negative (NEG) control construct pJM105?L1.3
(lacking a functional ORF2 gene) in the presence or absence (POS) of the
indicated APOBEC3 proteins.
Inhibition of LINE-1 retrotransposition and HIV-1?Vif infectivity by
inhibitory activity of A3A and A3B. (A) This assay measures the ability of the
in bacteria (40). Plasmids encoding the indicated proteins were introduced
into E. coli, and their expression was activated by using isopropyl ?-D-
thiogalactoside. The level of mutation induced by each protein was then
assessed by plating the bacteria on plates containing rifampicin and counting
the number of resistant colonies. (B) Effect of the indicated wild-type or
mutant A3A and A3B proteins on retrotransposition of human LINE-1 or the
murine LTR retrotransposon IAP. This analysis of IAP retrotransposition fre-
quency was performed as described in ref. 23. The average of three indepen-
dent experiments is shown.
Effect of mutations on the CDA activity and retrotransposition
www.pnas.org?cgi?doi?10.1073?pnas.0603313103 Bogerd et al.
we also measured their ability to inhibit the mobility of the
mouse LTR retrotransposon intracisternal A particle (IAP) and,
in the case of N-A3B and C-A3B, the infectivity of HIV-1
virions. We recently demonstrated that A3A is a potent inhibitor
of IAP retrotransposition and that inhibition of IAP by A3A
does not require an intact CDA active site (23). As shown in Fig.
8B, all of these mutant proteins were expressed at comparable
levels in transfected human cells.
The most important point to emerge from these studies is that
two of the mutant APOBEC3 proteins that lack any detectable
CDA activity (i.e., the full-length A3B mutant A3Bm2 and the
N-terminal 192-aa segment of A3B termed N-A3B) nevertheless
remain fully active against both LINE-1 and IAP (Fig. 3B).
are at least as active as full-length A3B in inhibiting LINE-1
retrotransposition, although both these ‘‘half-proteins’’ differ
from full-length A3B in being entirely inactive against HIV-
1?Vif (Fig. 2A).
Because the division of the A3G ORF into analogous N-
terminal (N-A3G) and C-terminal (C-A3G) segments also re-
sulted in the production of stable half-proteins (Figs. 1A and 8
C and D), we also asked whether these truncated proteins were
biologically active. As expected, wild-type A3G and C-A3G
enhanced mutagenesis in bacteria to an equivalent degree,
whereas the N-A3G protein was inactive (Fig. 9A, which is
published as supporting information on the PNAS web site).
However, neither N-A3G nor C-A3G could inhibit either
LINE-1 retrotransposition or HIV-1 infectivity (Fig. 9B). The
inability of both C-A3G (Fig. 9B) and wild-type A3C (Fig. 2A)
to effectively inhibit LINE-1 retrotransposition argues that
expression of an enzymatically active CDA in the nucleus is not
sufficient to inhibit LINE-1 mobility.
Although the above data argue that CDA activity is neither
necessary nor sufficient for effective inhibition of LINE-1 ret-
rotransposition by an APOBEC3 protein, it appears that an
intact CDA consensus site nevertheless plays an important role
in mediating this effect. In particular, introduction of the E68Q
mutation into N-A3B, a protein that already lacks detectable
CDA enzyme activity (Fig. 3A), nevertheless alleviated inhibi-
tion of LINE-1 retrotransposition (Fig. 3B), although inhibition
of IAP retrotransposition was less affected. Similarly, mutation
of the CDA active sites that are present in A3A (A3Am) and
C-A3B (C-A3Bm2) also strongly attenuated the observed inhi-
bition of LINE-1 mobility, although, again, inhibition of IAP was
less affected. The N-terminal CDA consensus site of A3G is not
enzymatically active yet appears to be required for efficient
packaging of A3G into HIV-1 virion particles (38, 39). Similarly,
it appears that an intact CDA consensus site plays an important
role in mediating the inhibition of LINE-1 retrotransposition by
A3A and A3B, perhaps by allowing a specific interaction with
LINE-1 ribonucleoprotein complexes, although enzymatic ac-
tivity per se is not important.
Inhibition of Alu Retrotransposition by Human A3A and A3B. Alu
elements represent a highly abundant family of human short
interspersed elements and constitute ?11% of the entire human
genome (5). Retrotransposition of Alu elements is mediated by
the LINE-1 ORF2 protein (4), which has reverse transcriptase
and endonuclease activities (35, 42) but does not require the
LINE-1 ORF1 RNA-binding protein (4).
To test whether human APOBEC3 proteins can inhibit Alu
mobility, we cotransfected HeLa cells with a previously de-
scribed Alu construct tagged with a retrotransposition indicator
cassette (4) together with a plasmid expressing either full-length
wild-type LINE-1 or only LINE-1 ORF2p. Finally, the cells were
also cotransfected with plasmids expressing A3A, A3B, A3C, or
the A3Am mutant. A3A and A3B both acted as potent inhibitors
of Alu retrotransposition regardless of whether both ORF1p and
ORF2p, or only ORF2, were coexpressed (Fig. 4; see also Fig.
10, which is published as supporting information on the PNAS
web site). As in the case of LINE-1 (Figs. 2 and 3B), the A3Am
mutant bearing an inactive CDA consensus sequence failed to
a moderate inhibitory effect. We therefore conclude that A3A
and A3B can inhibit retrotransposition of both LINE-1 and
Alu and that, at least in the latter case, this inhibition does not
involve an interaction with the LINE-1 ORF1 protein. Sequence
analysis of Alu elements that had retrotransposed in the pres-
of C-to-T or G-to-A mutagenesis when compared with control
cultures (Fig. 11, which is published as supporting information
on the PNAS web site). These data suggest that inhibition of Alu
retrotransposition, like inhibition of LINE-1 retrotransposition,
does not result from editing of reverse transcripts.
A3B mRNA Is Expressed During Early Human Development. Although
LINE-1 and Alu can retrotranspose in some somatic tissues, to
propagate they must retrotranspose in germ cells and?or during
the earliest stages in human embryonic development (1, 2).
Because mice do not encode A3A and A3B homologs (17),
inhibition of LINE-1 and Alu mobility can be investigated only
by using human tissue samples. Moreover, because an antibody
specific for A3A or A3B does not exist yet, analysis of A3A or
A3B expression can currently be performed only at the mRNA
this mRNA is present at a low to very low level in a wide range
of human somatic tissues (17, 18, 29). In contrast, A3A mRNA
expression has been reported in peripheral blood lymphocytes
(PBLs) but not elsewhere (17). These data were confirmed by
RT-PCR analysis, which showed a low level of A3B mRNA
testis, whereas A3A mRNA was detected only in PBLs and in
spleen (Fig. 12, which is published as supporting information on
the PNAS web site).
We also analyzed mRNA samples derived from three distinct
undifferentiated hES cell lines (H9p47, BG01p53, and hSF-
6p48). RT-PCR analysis detected both A3B mRNA and LINE-1
mRNA in these hES cell lines, although A3A mRNA was not
detected. We therefore conclude that A3B mRNA is expressed
in human tissues, including very early embryonic tissues, where
novel LINE-1 and Alu retrotransposition events could lead to
new heritable insertions.
fected with an Alu construct tagged with a retrotransposition indicator
cassette, an APOBEC3 expression plasmid, or a control plasmid (pK??-arr) and
days after transfection, cells were subjected to G418 selection, and neo-
resistant colonies were stained and counted 12 days later. Data are presented
relative to the culture cotransfected with pK??-arrestin (Control), which was
with standard deviation indicated.
A3A and A3B inhibit Alu retrotransposition. HeLa cells were cotrans-
Bogerd et al.
June 6, 2006 ?
vol. 103 ?
no. 23 ?
Although the human APOBEC3 gene family is undergoing rapid
adaptive evolution, the driving force(s) behind this evolution has
remained unclear (20, 21). Given that the only known function
of the APOBEC3 proteins is as inhibitors of retroelements, the
most likely sources of positive selection for novel APOBEC3
sequences and activities are exogenous retroviruses, endogenous
retroviruses, the closely related LTR retrotransposons, and,
finally, non-LTR retrotransposons.
A3G was initially identified as a potent inhibitor of Vif-
deficient HIV-1 (13), and at least four human APOBEC3 gene
products (A3G, A3B, A3F, and A3C) have subsequently been
shown to inhibit various non-human exogenous retroviruses as
well as hepatitis B virus (6). The APOBEC3 proteins may
therefore play an important role in preventing the zoonotic
transmission of animal retroviruses into the human population,
but this hypothesis is difficult to test.
The second possibility (i.e., that APOBEC3 proteins play a
role in controlling endogenous human retroviruses and LTR
retrotransposons) seems unlikely because the human genome
does not contain any known biologically active LTR retrotrans-
posons and few if any functional endogenous retroviruses (1, 5).
Therefore, although A3A, A3B, and A3G can inhibit the mo-
bility of LTR retrotransposons of murine origin (23, 24), human
endogenous retroviruses and LTR retrotransposons seem un-
likely of being capable of exerting significant selective pressure,
at least at this stage in human and primate evolution.
Unlike LTR retrotransposons, the human genome does con-
tain a substantial number of biologically active non-LTR retro-
transposons, and novel germ-line non-LTR retrotransposition
events are believed to occur in ?10% of all humans (7–10). As
a result, non-LTR retrotransposons, of which the most common
are LINE-1 and Alu, have accumulated over time to comprise
?30% of the entire human genome and continue to influence
the evolution of that genome (5). The question then is: Do the
human APOBEC3 proteins play a role in restricting non-LTR
retrotransposon mobility to the low level that is currently seen?
In this study, we present evidence arguing that at least two
members of the human APOBEC3 protein family, A3A and
A3B, can indeed act as effective inhibitors of LINE-1 and Alu
retrotransposition (Figs. 2 and 4). Moreover, at least in the case
of A3B, mRNA encoding this protein is readily detectable in
both hES cells (Fig. 5) and in human testis and ovary (Fig. 12),
thus suggesting that the A3B protein is expressed in the right
places to exert a physiologically relevant inhibitory effect on the
appearance of novel heritable insertions of LINE-1 or Alu.
The APOBEC3 proteins are CDAs, and inhibition of exoge-
nous retrovirus replication is associated with hypermutation of
retroviral proviruses (6, 14–16). However, recent data demon-
strate that certain mutants of A3G or A3A retain the ability to
inhibit HIV-1 or LTR retrotransposon replication, respectively,
even in the absence of a functional CDA active site (23, 38).
Here, we report that inhibition of LINE-1 or Alu retrotranspo-
sition by A3A or A3B does not result in detectable hypermuta-
tion (Figs. 7 and 11), and we have also identified A3B mutants
that are fully able to inhibit LINE-1 retrotransposition yet are
inactive as CDAs (Fig. 3). The mechanism(s) underlying inhi-
bition of LINE-1 mobility by A3B in the absence of editing, or
the inhibition of HIV-1 infectivity by enzymatically inactive
forms of A3G, currently remains unclear. However, the obser-
vation that A3A and A3B are effective inhibitors of Alu retro-
transposition mediated solely by the LINE-1 ORF2 protein (i.e.,
in the absence of the LINE-1 ORF1 RNA-binding protein)
suggests that this inhibition is not mediated by specific recruit-
ment of A3A or A3B by a protein that is functionally equivalent
to the retroviral nucleocapsid domain of Gag. This result is
surprising, because this domain of Gag has been shown to be
critical for the recruitment of APOBEC3 proteins into retroviral
virion particles, where they then exert their inhibitory effect (6).
It remains possible that an unknown cellular RNA-binding
protein(s) may play a role in mediating both Alu retrotranspo-
sition and APOBEC3 recruitment.
Although the A3A, A3B, and, to a lesser extent, A3C proteins
inhibit both LINE-1 and Alu retrotransposition (Figs. 2 and 4),
A3G and A3F have little or no effect on LINE-1 mobility,
although these proteins do function as potent inhibitors of
Vif-deficient HIV-1 (Fig. 2). This distinction correlates with the
observation that A3A, A3B, and A3C can enter the cell nucleus,
where LINE-1 reverse transcription occurs, whereas A3F and
A3G are restricted to the cytoplasm (Fig. 1). In the case of A3A
and A3C, nuclear entry appears to be due to passive diffusion,
but A3B clearly contains both a NLS and a nuclear export signal
(NES), thus implying that it functions in both the nucleus and
cytoplasm of expressing cells. Once the NES and NLS in A3B
have been defined, it will be of interest to see whether these
protein sorting signals play a role in mediating the inhibition of
LINE-1 mobility and HIV-1 infectivity that are characteristic of
A3B (Fig. 2A). In the interim, it is interesting to speculate about
how cells avoid the problem of random mutagenesis of their
DNA genome by nuclear APOBEC3 proteins.
Materials and Methods
Molecular Clones. The LINE-1 retrotransposition indicator con-
structs pJM101?L1.3 and pJM105?L1.3, the wild-type LINE-1
expression vector pJM101?L1.3?neo, and the LINE-1 ORF2
expression plasmid pCep 5?UTR ORF2?neo are described in
refs. 32, 33, and 44. Also previously described are the Alu
retrotransposition indicator construct AluneoTet(4) and the
HIV-1 proviral indicator construct pHIV-Luc?Vif (41). pK-
based expression plasmids expressing C-terminally HA-tagged
?-arrestin, A3A, A3B, A3C, A3F, and A3G are described in ref.
23. A3B and A3G cDNAs encoding C-terminally HA-tagged
N-A3B (amino acids 1–192), C-A3B (amino acids 193–382),
N-A3G (amino acids 1–196), and C-A3G (amino acids 197–384)
used to assess the expression of LINE-1, Sox2, A3A, and A3B mRNA in three
undifferentiated hES cell lines (H9p47, BG01p53, and hSF-6p48). A positive
control for the RT-PCR is shown beside the gel panel for A3A. The procedures
used for hES cell culture and RT-PCR analyses are described in Supporting
Analysis of A3A and A3B mRNA expression in hES cells. RT-PCR was
www.pnas.org?cgi?doi?10.1073?pnas.0603313103Bogerd et al.
were generated by PCR and cloned into the pK vector. All A3A
and A3B point mutants were generated by site-directed mu-
tagenesis and verified by DNA sequencing.
Bacterial Mutator Assay. APOBEC3 cDNAs (wild type and mu-
tant), including the C-terminal HA tag, were excised from the
relevant pK-based plasmid by cleavage with Asp-718 and XhoI
and subcloned into Asp718 and SalI sites present in the bacterial
expression plasmid pTrc99A (AP Biosciences, Piscataway, NJ).
The uracil DNA glycosylase-deficient E. coli strain BW310 (40)
was transformed with the pTrc99A parental plasmid and the
various wild-type and mutant A3A and A3B derivatives. Trans-
formed bacteria were then selected overnight on LB plates
containing ampicillin. Twenty colonies were pooled into 2 ml of
LB plus ampicillin plus 1 mM isopropyl ?-D-thiogalactoside
(IPTG), and cultures were grown overnight at 37°C. One hun-
dred microliters of the saturated culture was then plated on LB
plates containing 100 ?g?ml rifampicin, and the total number of
rifRcolonies per plate was counted 24 h later. To verify protein
expression, 100 ?l of the saturated IPTG-induced culture was
lysed, subjected to gel electrophoresis, and analyzed by Western
blot as previously described.
Note. While this report was under review, Chen et al. (43) reported that
A3A can inhibit LINE-1 retrotransposition.
We thank Michael Malim (King’s College, London), Reuben Harris
(University of Minnesota, Minneapolis), Bryce Paschal (University of
Virginia, Charlottesville), and Thierry Heidmann (Institute Gustave
Roussy, Villejuif, France) for reagents and Brian Doehle for help with
the construction of mutants. This work was supported by National
Institutes of Health Grants AI65301 (to B.R.C.), GM60518 (to J.V.M.),
and GM69985 (to K.S.O.); Ministerio de Educacio ´n y Ciencia de
Espan ˜a?Fulbright Postdoctoral Grant EX-200300881 (to J.L.G.-P.); and
National Institutes of Health Michigan Predoctoral Training Grant
5T32GM07544 (to A.E.H.).
1. Moran, J. V. & Gilbert, N. (2002) in Mobile DNA II, eds. Craig, N., Craggie,
R., Gellert, M. & Lambowitz, A. (Am. Soc. Microbiol., Washington, DC), pp.
2. Moran, J. V., Holmes, S. E., Naas, T. P., DeBerardinis, R. J., Boeke, J. D. &
Kazazian, H. H., Jr. (1996) Cell 87, 917–927.
3. Batzer, M. A. & Deininger, P. L. (2002) Nat. Rev. Genet. 3, 370–379.
4. Dewannieux, M., Esnault, C. & Heidmann, T. (2003) Nat. Genet. 35, 41–48.
5. Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin,
J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., et al. (2001) Nature 409,
6. Cullen, B. R. (2006) J. Virol. 80, 1067–1076.
7. Sassaman, D. M., Dombroski, B. A., Moran, J. V., Kimberland, M. L., Naas,
T. P., DeBerardinis, R. J., Gabriel, A., Swergold, G. D. & Kazazian, H. H., Jr.
(1997) Nat. Genet. 16, 37–43.
8. Brouha, B., Schustak, J., Badge, R. M., Lutz-Prigge, S., Farley, A. H., Moran,
J. V. & Kazazian, H. H., Jr. (2003) Proc. Natl. Acad. Sci. USA 100, 5280–5285.
9. Kazazian, H. H., Jr. (1999) Nat. Genet. 22, 130.
10. Li, X., Scaringe, W. A., Hill, K. A., Roberts, S., Mengos, A., Careri, D., Pinto,
M. T., Kasper, C. K. & Sommer, S. S. (2001) Hum. Mutat. 17, 511–519.
11. Kazazian, H. H., Jr., & Moran, J. V. (1998) Nat. Genet. 19, 19–24.
12. Ostertag, E. M. & Kazazian, H. H., Jr. (2001) Annu. Rev. Genet. 35, 501–538.
13. Sheehy, A. M., Gaddis, N. C., Choi, J. D. & Malim, M. H. (2002) Nature 418,
14. Mangeat, B., Turelli, P., Caron, G., Friedli, M., Perrin, L. & Trono, D. (2003)
Nature 424, 99–103.
15. Zhang, H., Yang, B., Pomerantz, R. J., Zhang, C., Arunachalam, S. C. & Gao,
L. (2003) Nature 424, 94–98.
16. Harris, R. S., Bishop, K. N., Sheehy, A. M., Craig, H. M., Petersen-Mahrt, S. K.,
Watt, I. N., Neuberger, M. S. & Malim, M. H. (2003) Cell 113, 803–809.
17. Jarmuz, A., Chester, A., Bayliss, J., Gisbourne, J., Dunham, I., Scott, J. &
Navaratnam, N. (2002) Genomics 79, 285–296.
18. Yu, Q., Chen, D., Ko ¨nig, R., Mariani, R., Unutmaz, D. & Landau, N. R. (2004)
J. Biol. Chem. 279, 53379–53386.
19. Bishop, K. N., Holmes, R. K., Sheehy, A. M., Davidson, N. O., Cho, S.-J. &
Malim, M. H. (2004) Curr. Biol. 14, 1392–1396.
20. Sawyer, S. L., Emerman, M. & Malik, H. S. (2004) PLoS Biol. 2, 1278–1285.
21. Zhang, J. & Webb, D. M. (2004) Hum. Mol. Genet. 13, 1785–1791.
22. Dutko, J. A., Scha ¨fer, A., Kenny, A. E., Cullen, B. R. & Curcio, M. J. (2005)
Curr. Biol. 15, 661–666.
23. Bogerd, H. P., Wiegand, H. L., Doehle, B. P., Lueders, K. K. & Cullen, B. R.
(2006) Nucleic Acids Res. 34, 89–95.
24. Esnault, C., Heidmann, O., Delebecque, F., Dewannieux, M., Ribet, D., Hance,
A. J., Heidmann, T. & Schwartz, O. (2005) Nature 433, 430–433.
25. Turelli, P., Vianin, S. & Trono, D. (2004) J. Biol. Chem. 279, 43371–43373.
26. Feng, Q., Schumann, G. & Boeke, J. D. (1998) Proc. Natl. Acad. Sci. USA 95,
27. Cost, G. J., Feng, Q., Jacquier, A. & Boeke, J. D. (2002) EMBO J. 21,
28. Go ¨rlich, D. & Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607–660.
29. Doehle, B. P., Scha ¨fer, A. & Cullen, B. R. (2005) Virology 339, 281–288.
30. Doehle,B.P.,Scha ¨fer,A.,Wiegand,H.L.,Bogerd,H.P.&Cullen,B.R.(2005)
J. Virol. 79, 8201–8207.
31. Black, B. E., Le ´vesque, L., Holaska, J. M., Wood, T. C. & Paschal, B. M. (1999)
Mol. Cell. Biol. 19, 8616–8624.
32. Wei, W., Morrish, T. A., Alisch, R. S. & Moran, J. V. (2000) Anal. Biochem.
33. Wei, W., Gilbert, N., Ooi, S. L., Lawler, J. F., Ostertag, E. M., Kazazian, H. H.,
Boeke, J. D. & Moran, J. V. (2001) Mol. Cell. Biol. 21, 1429–1439.
34. Freeman, J. D., Goodchild, N. L. & Mager, D. L. (1994) BioTechniques 17, 46,
35. Mathias, S. L., Scott, A. F., Kazazian, H. H., Jr., Boeke, J. D. & Gabriel, A.
(1991) Science 254, 1808–1810.
36. Turelli, P., Mangeat, B., Jost, S., Vianin, S. & Trono, D. (2004) Science 303,
37. Rosier, C., Kock, J., Kann, M., Malim, M. H., Blum, H. E., Baumert, T. F. &
von Weizsacker, F. (2005) Hepatology 42, 301–309.
38. Newman, E. N. C., Holmes, R. K., Craig, H. M., Klein, K. C., Lingappa, J. R.,
Malim, M. H. & Sheehy, A. M. (2005) Curr. Biol. 15, 166–170.
39. Navarro, F., Bollman, B., Chen, H., Ko ¨nig, R., Yu, Q., Chiles, K. & Landau,
N. R. (2005) Virology 333, 374–386.
40. Hache ´, G., Liddament, M. T. & Harris, R. S. (2005) J. Biol. Chem. 280,
41. Wiegand, H. L., Doehle, B. P., Bogerd, H. P. & Cullen, B. R. (2004) EMBO
J. 23, 2451–2458.
42. Feng, Q., Moran, J. V., Kazazian, H. H., Jr., & Boeke, J. D. (1996) Cell 87,
43. Chen, H., Lilley, C. E., Yu, Q., Lee, D. V., Chou, J., Narvaiza, I., Landau, N. R.
& Weitzman, M. D. (2006) Curr. Biol. 16, 480–485.
44. Alisch, R. S., Garcia-Perez, J. L., Muotri, A. R., Gage, F. H. & Moran, J. V.
(2006) Genes Dev. 20, 210–224.
45. Wiegand, H. L., Coburn, G. A., Zeng, Y., Kang, Y., Bogerd, H. P. & Cullen,
B. R. (2002) Mol. Cell. Biol. 22, 245–256.
Bogerd et al.
June 6, 2006 ?
vol. 103 ?
no. 23 ?