JOURNAL OF VIROLOGY, June 2006, p. 5984–5991
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 12
Mutational Alteration of Human Immunodeficiency Virus Type 1 Vif
Allows for Functional Interaction with Nonhuman Primate APOBEC3G†
Ba ¨rbel Schro ¨felbauer,1,3Tilo Senger,1,4Gerard Manning,2and Nathaniel R. Landau1*
Infectious Disease Laboratory1and Newman Center for Bioinformatics,2The Salk Institute for Biological Studies, La Jolla,
California 92037; Department of Biotechnology, Institute of Applied Microbiology, University of Natural Resources
and Applied Life Sciences, A-1180 Vienna, Austria3; and Technical University Carolo-Wilhelmina of
Braunschweig, Braunschweig, Germany4
Received 23 February 2006/Accepted 5 April 2006
Human APOBEC3F (hA3F) and APOBEC3G (hA3G) are antiretroviral cytidine deaminases that can be
encapsidated during virus assembly to catalyze C3U deamination of the viral reverse transcripts in the next
round of infection. Lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency
virus (SIV) have evolved the accessory protein Vif to induce their degradation before packaging. HIV type 1
(HIV-1) Vif counteracts hA3G but not rhesus macaque APOBEC3G (rhA3G) or African green monkey (AGM)
APOBEC3G (agmA3G) because of a failure to bind the nonhuman primate proteins. The species specificity of
the interaction is controlled by amino acid 128, which is aspartate in hA3G and lysine in rhA3G. With the
objective of overcoming this species restriction, mutations were introduced into HIV-1 Vif at amino acid
positions that differed in charge between HIV-1 Vif and SIV Vif. The mutant proteins were tested for the ability
to counteract hA3G, rhA3G, and agmA3G. Alteration of the conserved sequence at positions 14 to 17 from
DRMR to SERQ, which is the sequence in AGM Vif, caused HIV-1 Vif to functionally interact with rhA3G and
agmA3G. Mutation of three residues to the sequence SEMQ allowed interaction with rhA3G. SEMQ Vif also
counteracted D128K mutant hA3G and wild-type hA3G. Introduction of the sequence into an infectious
molecular HIV-1 clone allowed the virus to replicate productively in human cells that expressed rhA3G or
hA3G. These findings provide insight into the interaction of Vif with A3G and are a step toward the develop-
ment of a novel primate model for AIDS.
Several nonhuman primate models for AIDS have been
used to provide insight into human immunodeficiency virus
(HIV) pathogenesis in humans. In particular, simian immuno-
deficiency virus of macaques (SIVmac) and chimeric SIV-HIV
induce AIDS-like symptoms in rhesus macaques through
pathogenic mechanisms that mimic those of HIV type 1
(HIV-1) (7). HIV-1, however, does not infect rhesus macaques
or nonhuman primates such as the African green monkey
(AGM) (1, 10–12, 19, 23). This species restriction is caused by
at least two blocks to HIV-1 replication in nonhuman primate
T cells and macrophages: a defect in the particle infectivity of
virions that are released which is mediated by host APOBEC3
proteins and a postentry block that is mediated by Trim5? (25).
Lentiviruses such as HIV-1 and SIV encode Vif, an acces-
sory protein that counteracts the antiviral activity of the APO-
BEC3 family of cytidine deaminases (9). Human APOBEC3G
(hA3G) was identified initially (22), and subsequently addi-
tional family members, including human APOBEC3B, human
APOBEC3C, and human APOBEC3F (hA3F), were found to
be active (14, 27, 34). In cells infected with HIV-1 with vif
deleted (?vif), hA3G and hA3F are packaged into assembling
virions. In the next round of infection, the packaged enzymes
deaminate the newly synthesized minus-strand cDNA, result-
ing in plus-strand G3A mutations (8, 15, 17, 30, 33). In cells
infected with wild-type HIV-1, Vif binds to hA3G and hA3F,
forming a complex with a Cul5-based E3 ligase. The A3G
proteins are then ubiquitinated and degraded by proteasomes
(18, 31). Human APOBEC3B and human APOBEC3C are
weakly active against HIV-1 but are potent inhibitors of SIV
(2, 29). Other elements targeted by APOBEC3 family mem-
bers include hepatitis B, endogenous murine long terminal
repeat and non-long terminal repeat retroelements, and yeast
retrotransposons (4–6, 26).
The interaction of Vif with hA3G is species specific (17).
HIV-1 Vif binds to hA3G but not to rhesus macaque
APOBEC3G (rhA3G) or AGM APOBEC3G (agmA3G).
Conversely, SIV of AGMs (SIVagm) Vif binds to agmA3G but
not to hA3G. SIVmac Vif is more permissive, binding to
rhA3G, agmA3G, and hA3G. The determinant on hA3G that
controls the species specificity of its interaction with Vif was
mapped on a panel of hA3G-agmA3G chimeras. The specific-
ity mapped to amino acid 128, which is Lys in hA3G but Asp
in agmA3G (3, 16, 21, 28). Exchange of the Lys and Asp
residues reverses the species specificity for binding to Vif.
SIVagm Vif binds to D128K mutant hA3G but not to K128D
mutant agmA3G. Conversely, HIV-1 Vif binds to K128D mu-
tant agmA3G but not to D128K mutant hA3G. Modeling on
the Escherichia coli cytidine deaminase predicts that amino
acid 128 lies on an exposed loop that is positioned away from
the enzyme’s active site. The exchange does not affect the
catalytic activity of the enzyme (21).
The objective of this study was to generate a genetically
altered HIV-1 that could functionally interact with nonhuman
* Corresponding author. Mailing address: Infectious Disease Labo-
ratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines
Rd., La Jolla, CA 92037. Phone: (858) 453-4100. Fax: (858) 554-0341.
† Supplemental material for this article may be found at http://jvi
primate APOBEC3 to counteract its antiviral activity. The
approach taken was to identify amino acids in HIV-1 Vif that
contribute to it species specificity and to alter these to resem-
ble the sequence in SIV Vif. We found that a four-amino-acid
sequence near the amino terminus of Vif which differs in
charge between HIV-1 and SIV is important in determining
the species specificity of the interaction with A3G. Alteration
of the sequence resulted in a Vif that was able to counteract
rhA3G and hA3G and allowed productive replication in hu-
man cells that stably expressed the deaminases. The mutation
also allowed interaction with D128K mutant hA3G. These
findings provide an important step toward the development of
a primate model for HIV-1 replication.
MATERIALS AND METHODS
Vif expression vectors. A codon-optimized HIV-1 vif gene was assembled from
synthetic oligonucleotides and amplified with a forward primer containing an
EcoRI site and a reverse primer encoding a V5 epitope tag and an XhoI site (see
Fig. S1 in the supplemental material). The PCR product was cloned into
pcDNA3.1 at the EcoRI and XhoI sites to generate pcVif-Co. Mutations in Vif
were generated in pcVif-Co by overlapping PCR and confirmed by nucleotide
sequencing. Expression vector pcVifmac was constructed by amplification of the
Vif open reading frame (ORF) from SIVmac239 with primers containing EcoRI
and XhoI sites and encoding a carboxy-terminal V5 epitope tag. The PCR
product was cleaved and cloned into pcDNA3.1 at the EcoRI and XhoI sites.
Viral plasmids. Single-cycle luciferase reporter viruses pNL-Luc-E?R?, pNL-
Luc-E?R??vif, and pSIVmac-Luc-E?R?and replication-competent viruses
NL-R?and NL-R?V?have been previously described (17). The SEMQ muta-
tion was introduced into pNL-Luc-E?R?and NL43-R?by overlapping PCR in
the fragment spanning the region between sites AgeI and PflmI (nucleotides
3485 to 5303) (data not shown). pNL4-3(Vifmac) is a ?vif NL4-3 Vpr?plasmid
into which the SIVmac239 Vif ORF has been inserted in the nef gene. The
plasmid was constructed by amplifying the SIVmac239 Vif ORF with primers
containing NotI and XhoI restriction sites for cloning into nef at the same
position as in NL-Luc.
Luciferase reporter virus assay. Vif function was analyzed as previously de-
scribed (17, 21). Briefly, vesicular stomatitis virus G (VSV-G)-pseudotyped lu-
ciferase reporter viruses were produced in 293T cells by cotransfection of 2 ?g of
pNL-Luc-E?R??vif, 1 ?g of pcVif-Co.V5, 0.5 ?g of an hA3G or rhA3G expres-
sion vector, and 0.5 ?g of pcVSV-G. Virus was harvested 48 h posttransfection,
and 1 ng of p24 was used to infect HOS.T4 cells in triplicate. Three days later,
intracellular luciferase activity was measured with Luc-Lite Plus reagent (Pack-
ard) in a Topcount luminometer (Perkin-Elmer). The data are presented as
mean counts per second ? the standard deviation. Vif expression in transfected
cells was verified by immunoblot assay and probing with anti-V5 monoclonal
antibody (MAb; Invitrogen). For titration experiments, 2 ?g of pNL-Luc-E?R?,
pNL-Luc-E?R??vif, pNL-Luc-E?R?Vif SEMQ, or pSIVmac-Luc-E?R?was
cotransfected with indicated amounts of rhA3F, hA3G, rhA3G, or D128K mu-
tant hA3G and pcVSV-G. Viruses were normalized for p24 or p27, and 1 ng was
used to measure their infectivity as described above.
Encapsidation of A3G. Virions were produced by cotransfection of 293T cells
with 2 ?g of pNL-Luc-E?R?, pNL-Luc-E?R??vif, pNL-Luc-E?R?Vif SEMQ
with or without 1 ?g of pcSIVmac Vif.V5, and 1.0 ?g of hA3G.HA (where HA
is hemagglutinin), rhA3G.HA, or D128K mutant hA3G.HA. The cells and su-
pernatant were harvested 2 days posttransfection, and virus was concentrated by
ultracentrifugation through 20% sucrose at 40,000 rpm for 1 h. A3G was de-
tected in 10 ng of p24 and 20 ?g of cell lysates by an immunoblot assay and
probing with an anti-HA MAb. The blots were stripped and reprobed with an
anti-Vif MAb, an anti-V5 MAb, or AIDS patient serum. The blots were also
probed with an antitubulin MAb to control for equivalent loading.
Virus replication kinetics. The kinetics of viral replication was determined
with HOS.T4.X4 cells that stably expressed hA3G.HA or rhA3G.HA.
HOS.T4.X4 cells were infected with a VSV-G-pseudotyped pBABE-neo retro-
viral vector, and cells were selected with 0.5 mg/ml G418. Resistant clones were
isolated and expanded, and rhA3G expression was confirmed by Western blot
analysis. Cell clones were infected with 10 ng of p24 from wild-type, ?vif, or Vif
SEMQ replication-competent virus, and virus growth was measured over 15 days
by p24 enzyme-linked immunosorbent assay.
Alteration of a three-amino-acid sequence in HIV-1 Vif al-
lows interaction with rhA3G. A functional interaction of
HIV-1 Vif with hA3G requires amino acid 128 of hA3G to be
negative or neutral. Conversely, interaction of SIVagm Vif
with agmA3G requires a positive charge at amino acid 128. We
hypothesized that these charge requirements may result from
the interaction of complementary charged amino acids in Vif
and A3G. D128 in hA3G could interact with a positive charge
in HIV-1 Vif, and K128 in agmA3G could interact with a
negative charge in SIV Vif. To search for potential comple-
mentary amino acids in Vif that might interact with A3G
amino acid 128, we searched known viral sequences to identify
amino acid residues of Vif that were positively charged in
HIV-1 and negative in SIV. In the SIVagm Vif sequences of
the Los Alamos National Laboratory sequence database, there
are seven conserved, negatively charged positions (E4, E17,
E102, E104, D109, D/E122, and E215). Only two of the cor-
responding amino acids in HIV-1 Vif are positively charged
(R4 and R15, corresponding to SIVagm Vif E4 and E17, re-
spectively). HIV-1 Vif R15 is flanked by a nearby positive
charge at R17, and these amino acids are conserved in HIV-1
Vif (R15 in ?99% of isolates, and position 17 is either R or K
in all sequenced viruses [82% R and 18% K]). We therefore
focused on amino acids 14 to 17, which in HIV-1 Vif are
DRMR. In SIVagm, positions 15 and 17 of Vif are neutral or
negatively charged (Fig. 1A).
To test the role of amino acids 4 and 14 to 17 of HIV-1 Vif
in determining the species specificity of the interaction with
A3G, mutant Vif expression vectors were generated and their
function was tested in a single-cycle infection assay. Single-
cycle viruses were generated by cotransfection of 293T cells
with a ?vif HIV-1 luciferase reporter virus plasmid and a
mutant or wild-type pcVif-HA expression vector. The pcVif
plasmids expressed a Vif ORF that was codon optimized over
its entire length to increase expression in the triple transfection
(data not shown). A VSV-G expression vector was included in
the transfection to generate infectious pseudotyped virus. The
results of this analysis showed that a swap that included the R4
sequence (MEEEKR) maintained function against hA3G but
did not counteract rhA3G or agmA3G. In contrast, the swap of
the four-amino-acid sequence DRMR at positions 14 to 17 in
the SERQ mutant (changes are in bold) was active against
hA3G and rhA3G and partially active against agmA3G (Fig.
1B). Further analysis within positions 14 to 17 showed that
changing only three of the four amino acids in the SEMQ
mutant allowed interaction of HIV-1 Vif with rhA3G but not
with agmA3G. Mutation of R14E and R17E to DEME was not
sufficient to alter the interaction with A3G. Insertion of an E at
position 17 in DRMER was also insufficient. Immunoblot
analysis of the cell lysates showed that the mutant proteins
were expressed comparably to wild-type HIV-1 Vif (Fig. 1B,
bottom). These findings suggest that positions 14 to 17 of
HIV-1 Vif can influence the species specificity of the inter-
action with hA3G.
The role of this region in the interaction with rhA3G was
tested with additional HIV-1 Vif mutants with changes at po-
sitions 14 to 17. Point mutations at each of the three positions
(D14S, R15E, and R17Q) were tested (Fig. 2A). R15E failed
VOL. 80, 2006 INTERACTION OF HIV-1 Vif WITH PRIMATE APOBEC3G5985
to function on hA3G or rhA3G, while D14S and R15E were
active against hA3G but not against rhA3G. Thus, single mu-
tations were not sufficient to change the species specificity of
the interaction. SEMR, DEMQ, and SRMQ double mutants
were also tested (Fig. 2B). All double mutants remained at
least in part active against hA3G. Only DEMQ was partially
active on rhA3G. Immunoblot analysis confirmed the equiva-
lent expression of each of the mutant Vif proteins (Fig. 2A and
FIG. 1. A change of three amino acids in HIV-1 Vif allows functional interaction with rhA3G. (A) Alignment of HIV-1, SIVagm, and SIVmac
Vif sequences. (B) Vif function of the indicated constructs was determined in a luciferase reporter virus assay by cotransfection of a ?vif mutant
HIV-1 luciferase reporter together with the respective Vif construct, empty vector (no APO), hA3G (left part), rhA3G (middle part), or agmA3G
(right part) and pcVSV-G. The infectivity of the viruses was determined by infection of HOS.T4 cells with 1 ng of p24. The data are the averages
of triplicates with the indicated standard deviations. cps, counts per second.
FIG. 2. The charge of the amino acids in SEMQ determines the ability to interact with rhA3G. (A, B) The effects of single (A) and double
(B) mutations in the HIV-1 Vif DRMR sequence were tested against hA3G (top) and rhA3G (bottom) as described in the legend to Fig. 1. (C) The
functional interaction of mutant HIV-1 Vif proteins containing charge changes with hA3G (top) and rhA3G was tested on ?vif mutant HIV-1
luciferase reporters. The data are representative of three independent repetitions. cps, counts per second.
5986SCHRO ¨FELBAUER ET AL. J. VIROL.
B, bottom). Taken together, these findings mapped the mini-
mal required change to amino acids 14, 15, and 17. R15 and
R17 appear to be the most important.
The charge at positions 14 to 17 is a determinant of the
species specificity of the interaction with hA3G. To further
probe the role of positions 14 to 17, we tested the dependence
of charge on Vif function. The SDMQ and SHMQ mutants
tested the requirement for a negative charge at position 15.
The AEMQ mutant tested the importance of amino acid 14;
the SEMA and SEMN mutants tested position 17; and the
AAMA mutant tested positions 14, 15, and 17. All of the mu-
tants interacted at least partially with hA3G (Fig. 2C, upper
part). SDMQ but not SHMQ interacted with rhA3G, suggest-
ing a requirement for a negative charge at position 15 (Fig. 2C,
lower part). AEMQ, SEMA, and SEMN also interacted with
rhA3G, suggesting that the identity of amino acids 14 and 17 is
not critical but that they cannot be negative. AAMA was inac-
tive against rhA3G, further demonstrating the importance of a
negative charge at position 15. Taken together, these results
suggest that the charge at amino acids 14 to 17 influences the
interaction with rhA3G. Amino acid 15 must be negative, and
amino acid 17 must be neutral.
The SEMQ mutation allows interaction of HIV-1 Vif with
hA3G mutated at amino acid 128. The requirement for posi-
tive charges at positions 14 and 17 in HIV-1 Vif suggested an
interaction with a negative charge on hA3G. Because of its role
in determining the interaction with Vif, amino acid 128 was
considered a likely candidate interaction site on hA3G. To
determine whether this might be the case, we tested whether
SEMQ HIV-1 Vif would interact with D128K hA3G. As ex-
pected, DRMR did not. Interestingly, SEMQ interacted with
D128K mutant hA3G (Fig. 3A, right part). These results are
consistent with a direct interaction of hA3G D128 with DRMR
in Vif. In this analysis, the Vif mutations were introduced
directly into the HIV-1 reporter construct. Because of the
overlap with pol, the SEMQ mutation introduced three amino
acid changes in IN (see Fig. S1 in the supplemental material).
However, these did not interfere with the production of infec-
tious reporter virus. In addition, the analysis showed that the
Vif mutations were functional as expressed in cis in the context
of the virus, arguing that the previous results were not simply
the result of overexpression from the codon-optimized Vif
To determine the relative efficiency with which the SEMQ
mutation altered the Vif phenotype, the reporter viruses were
produced from 293T cells cotransfected with hA3G, rhA3G,
and D128K mutant hA3G expression vectors over a range of
plasmid ratios. Reporter viruses that expressed wild-type
FIG. 3. SEMQ mutant Vif in cis is active against hA3G, rhA3G, and D128K hA3G. (A) The infectivity of ?vif mutant HIV-1 Luc, wild-type
HIV-1 Luc, SEMQ mutant HIV-1 Vif Luc, and ?vif mutant HIV-1 Luc cotransfected with SIVmac Vif was tested against hA3G (left), rhA3G
(middle), and D128K mutant hA3G (right). (B) Infectivity of luciferase reporter viruses that encode mutant Vif. Wild-type and ?vif and SEMQ
mutant luciferase HIV-1 and SIVmac reporters were produced in cells cotransfected with increasing amounts of hA3G (left), rhA3G (middle), or
D128K hA3G (right). The infectivity of the virus produced in the absence of A3G was set to 100%, and the infectivity of mock-transfected cell
supernatant (4 ? 106to 6 ? 106and 200 to 2,000 cps [counts per second], respectively) was set to 0%.
VOL. 80, 2006 INTERACTION OF HIV-1 Vif WITH PRIMATE APOBEC3G5987
HIV-1 Vif, SEMQ Vif, and SIVmac Vif were all similarly
resistant to hA3G and differed from ?vif mutant virus, which
was sensitive (Fig. 3B, left part). A reporter virus that ex-
pressed HIV-1 Vif was inhibited by rhA3G (Fig. 3B, middle
part), in contrast to viruses that expressed SEMQ Vif and
SIVmac Vif, which were resistant. Viruses that expressed
HIV-1 Vif, SEMQ mutant HIV-1 Vif, and SIVmac Vif were all
similarly resistant to D128K mutant hA3G (Fig. 3B, right part).
Taken together, the results show that the SEMQ mutation
allows HIV-1 Vif to counteract rhA3G and D128K mutant
hA3G with an efficiency comparable to that of SIVmac Vif.
These results show that the SEMQ gene is functional as ex-
pressed by the virus and that the alteration to IN was not
SEMQ mutant Vif reduces A3G virion encapsidation. To
biochemically assess SEMQ mutant Vif function, its effect on
A3G virion encapsidation and steady-state intracellular level
was measured. 293T cells were transfected with ?vif mutant
or SEMQ mutant HIV-1 Vif and an A3G expression vector.
The virions were pelleted, and their A3G content was quanti-
tated by immunoblot analysis. hA3G was present in ?vif virions
but not in controls that lacked viral DNA or that expressed
SIVmac Vif or SEMQ mutant Vif (Fig. 4A, upper part). The
cell lysates showed that SIVmac Vif and SEMQ mutant Vif
reduced the steady-state level of hA3G (Fig. 4A, lower part).
HIV-1 Vif and SIVmac Vif were detected with anti-Vif and
anti-V5 epitope tag MAbs, respectively. SIVmac Vif appeared
to be present at higher levels, probably because it was supple-
mented in trans. HIV-1 Vif did not exclude rhA3G from virions
(Fig. 4B, upper part). In contrast, SIVmac Vif and SEMQ
mutant Vif effectively prevented the encapsidation of rhA3G.
SIVmac Vif and SEMQ mutant Vif correspondingly reduced
steady-state rhA3G levels in the cell lysates (Fig. 4B, lower
part). Similarly, HIV-1 Vif did not exclude D128K mutant
hA3G from virions but SIVmac and SEMQ mutant Vif were
effective and reduced the steady-state levels of D128K mutant
hA3G in the cell lysates (Fig. 4B, upper and lower parts).
SEMQ mutant Vif was expressed at amounts comparable to
wild-type Vif in each panel, suggesting that the effect was not
caused by differences in levels of Vif.
HIV-1 with the SEMQ mutation replicates in human cells
that stably express rhA3G. To test whether introduction of the
SEMQ mutation would allow HIV-1 to productively replicate
in cells that express rhA3G, the mutation was introduced into
replication-competent NL4-3. The SEMQ mutant NL4-3 virus,
along with control ?vif mutant and wild-type NL4-3, was used
to infect HOS.T4.X4 cells that stably expressed CD4/CXCR4
and hA3G or rhA3G or that lacked A3G. Clones of HOS cells
that expressed moderate (clone 8) or high (clone 5) levels of
rhA3G were used (Fig. 5A). Virus replication kinetics was
measured over 2 weeks. The three viruses replicated similarly
in cells that lacked A3G (Fig. 5B). On cells that express hA3G,
the wild-type and SEMQ mutant viruses replicated but the ?vif
mutant virus replicated poorly (Fig. 5C). On clone 8 and clone
5 rhA3G cells, the SEMQ mutant virus replicated efficiently,
unlike the ?vif mutant and wild-type viruses, which failed to
replicate (Fig. 5D). These results show that the SEMQ muta-
tion allows efficient replication of HIV-1 on cells that ex-
pressed relatively high levels of rhA3G.
rhA3F does not block HIV-1 replication. hA3F has previ-
ously been shown to block ?vif mutant HIV-1 infectivity in
single-round reporter virus assays (14, 27, 34). To test whether
A3F restricts HIV-1 in a species-specific manner similar to that
of A3G, we cloned rhA3F (see Fig. S2 in the supplemental
material) and tested its activity against wild-type, ?vif mutant,
and SEMQ mutant HIV-1 reporter viruses. As expected, hA3F
inhibited ?vif mutant but not wild-type HIV-1 (14, 27, 34). In
contrast, hA3F inhibited the SEMQ mutant virus. This sug-
gested that the SEMQ mutation caused Vif to lose its ability to
counteract hA3F (Fig. 6A). rhA3F inhibited wild-type HIV-1
and the SEMQ mutant. hA3F and rhA3F inhibited ?vif mutant
SIVmac, and this was partially overcome by SIVmac Vif, con-
sistent with a recent report by Zennou and Bieniasz (32) which
FIG. 4. SEMQ mutant Vif excludes hA3G, rhA3G, and D128K mutant hA3G from virions. (A) Encapsidation of hA3G (left), rhA3G (middle),
and D128K mutant hA3G (right) was tested in wild-type HIV-1 (lane 1), ?vif mutant HIV-1 (lane 2), ?vif mutant HIV-1 plus SIVmac Vif (lane
3), SEMQ mutant Vif HIV-1 (lane 4), and a no-virus control. Virus produced in cells cotransfected with A3G was pelleted by ultracentrifugation.
The viruses (top parts) and cell lysates (bottom parts) were analyzed by immunoblot assay and probing with an anti-HA MAb for A3G. Equal
loading of virions was confirmed by probing with AIDS patient serum. Expression of HIV-1 Vif was confirmed in the cell lysates by probing the
blot with an anti-HIV-1 Vif MAb, and that of SIVmac Vif was confirmed by probing the blot with an anti-V5 MAb. The blot was probed with an
antitubulin MAb to confirm equal loading.
5988SCHRO ¨FELBAUER ET AL.J. VIROL.
showed that SIVmac Vif was partially active against rhA3F.
Our data suggested that the interaction between Vif and A3F
is species specific and that SEMQ mutant Vif fails to interact
To determine whether rhA3F blocks the replication of
HIV-1, we infected HOS cells that stably express CD4/CXCR4
and rhA3F with replication-competent wild-type, ?vif mutant,
or SEMQ mutant NL4-3 or NL4-3(Vifmac), an HIV-1 strain
that contains an engineered SIVmacvif gene in the nef position.
The HOS cells expressed moderate (C1) or high (C4) levels of
rhA3F (Fig. 6B). All viruses replicated well in the parental
HOS.CD4.CXCR4 cell line (Fig. 6B). Surprisingly, intermedi-
ate and high levels of rhA3F had no effect on the replication
kinetics. Some differences in peak p24 production were evident
among the HOS cell clones, but these were caused by differ-
ences in coreceptor expression (data not shown). Taken to-
gether, these data demonstrate that although rhA3F is active
against HIV-1 in the single-cycle reporter virus assay, it does
not block virus replication under more physiological condi-
We report here progress in addressing the APOBEC3 block
to HIV-1 replication in nonhuman primate cells. Changing the
sequence DRMR at positions 14 to 17 to SEMQ, which re-
sembles the SIV sequence, allowed it to counteract the antivi-
ral activity of rhA3G and agmA3G. SEMQ mutant Vif induced
rhA3G degradation and prevented its encapsidation. The
SEMQ mutation allowed the production of infectious reporter
virus in cells cotransfected with rhA3G, and in replication-
competent HIV-1, it allowed productive replication in human
cells that stably expressed relatively high levels of hA3G or
rhA3G. The mutant Vif protein was inactive against rhA3F in
reporter virus assays, but HIV-1 was able to productively rep-
licate in cells that stably expressed rhA3F. The DRMR se-
quence is highly conserved in HIV Vif, consistent with a role as
an interaction site for A3G. D14, R15, and M16 are 99%
conserved in database HIV-1 sequences, while R17 is con-
served in 82% of isolates and replaced with Lys in 18% of
The DRMR sequence in HIV-1 Vif is important for in-
teraction with APOBEC3G but is not the only region that is
required for a functional interaction. The SEMQ mutant
gained the ability to interact with rhA3G but did not lose its
ability to interact with hA3G. This suggests that the inter-
action of Vif with APOBEC3G is stabilized by additional
binding sites. With the exception of Vif R15E, which lost its
function against hA3G, mutations in this region in general
had little effect on the interaction with hA3G. Thus, the
DRMR region is critical for the strength of binding with A3G but
is not the sole determinant of its interaction with hA3G. Con-
sistent with this finding, HIV-1 Vif binds with low affinity to
D128K hA3G in coimmunoprecipitation experiments (21).
This interaction was not sufficient to counteract the antiviral
A prediction of this work is that position 128 of A3G inter-
acts with Vif at amino acid 15 or 17. This is suggested by the
finding that replacement of R15 and R17 in HIV-1 Vif with
neutral or negatively charged residues allowed interaction with
rhA3G, which is positively charged at position 128. In addition,
mutations at R15 and R17 allowed HIV-1 Vif to interact with
D128K mutant hA3G, which contains only a single amino acid
difference from wild-type hA3G. The complementarity of the
changes in Vif and APOBEC3G suggests a direct electrostatic
interaction at these positions, although indirect effects on the
conformation of the proteins cannot be ruled out.
An earlier report by Kar et al. (13) described findings that
FIG. 5. Viral replication kinetics in cells expressing hA3G or rhA3G. (A) hA3G and rhA3G stably expressed by HOS.T4.X4 cells derived by
retroviral vector transduction were detected by an immunoblot assay and probing with an anti-HA MAb. The parental cells lack A3G. (B to E)
Replication kinetics of wild-type (WT), ?vif mutant, or SEMQ mutant Vif HIV-1 in HOS.T4.X4 cells expressing no A3G, hA3G, or different
amounts of rhA3G were determined. Supernatant capsid p24 was quantitated over 15 days.
VOL. 80, 2006 INTERACTION OF HIV-1 Vif WITH PRIMATE APOBEC3G5989
appear to be at odds with those presented here. In that work,
an HIV-1 vif gene was inserted into the nef position of ?vif
mutant SIVmac239. The chimeric virus replicated in rhesus
T cells, suggesting that HIV Vif-1 can substitute for Vif in
SIVmac239 and implying that HIV-1 Vif functionally interacts
with rhA3G. In contrast, we found that low levels of rhA3G
blocked HIV-1 replication. The explanation for this difference
is not obvious but could be related to the expression of Vif in
the nef position instead of its native context or could be related
to the use of primary rhesus T cells, which may express rela-
tively low levels of rhA3G.
SEMQ mutant Vif did not interact with rhA3F, probably
because the binding site on Vif for A3F is not identical to that
for A3G (24). This raised the question of whether rhA3F is an
important restriction factor for HIV-1 in nonhuman primate
cells. In humans, hA3F is expressed in T cells and is active
against HIV-1 (14, 27, 34). rhA3F is likely to be similarly
expressed in nonhuman primates. In single-cycle luciferase vi-
rus analyses, rhA3F inhibited HIV-1 and the inhibition was not
reversed by Vif. SIVmac was also inhibited by rhA3F, but this
was somewhat reversed by SIV Vif. In contrast, our analysis of
replication-competent virus on cells that stably expressed
rhA3F led to somewhat different conclusions. In this analysis,
which more closely mimics physiological conditions, rhA3F did
not inhibit HIV-1 replication. This suggests that rhA3F is not
an impediment to HIV-1 replication in nonhuman primate
cells. The SEMQ mutation in Vif may be sufficient to over-
come the APOBEC3 barrier to HIV-1 replication in nonhu-
man primates without the need for additional mutational al-
Trim5? remains as a barrier to HIV-1 replication in nonhu-
man primate cells. As a result, the SEMQ mutant does not
replicate in primary rhesus cells (data not shown). Owens et al.
(20) showed that various mutations in the HIV-1 capsid, most
FIG. 6. rhA3F does not block HIV-1 replication. (A) The antiviral effects of hA3F and rhA3F were tested in a single-cycle luciferase reporter
virus assay. Virus was produced by cotransfection of wild-type, ?vif mutant, or SEMQ mutant NL-Luc or wild-type (WT) or ?vif mutant SIVmac
Luc and the indicated A3F expression plasmid. HOS.T4 cells were infected with 1 ng of reporter virus, and intracellular luciferase activity was
measured 3 days postinfection. (B) Immunoblot analysis of V5-tagged rhA3F expression in HOS.T4.X4 cell clones or control cell clones (left).
Replication kinetics of wild-type, ?vif mutant, or SEMQ mutant NL4-3(Vifmac) and NL4-3(Vifmac), which expresses SIVmac239 Vif in the nef
position of NL4-3 on control and rhA3F-expressing HOS.T4.X4 cell clones. The cells expressed intermediate (C1) or high (C4) levels of rhA3F.
Supernatant p24 was measured over 2 weeks. cps, counts per second.
5990 SCHRO ¨FELBAUER ET AL.J. VIROL.
in the vicinity of the cyclophilin binding loop, partially allowed
the virus to escape rhTrim5?. The combination of these mu-
tations could be helpful in the establishment of a novel primate
model for AIDS.
We thank Hui Chen and Qin Yu for critical reading of the manu-
This work was funded by grants from the National Institutes of
Health (AI51686 and DA14494) and the American Foundation for
AIDS Research. N.R.L. is an Elizabeth Glaser Fellow of the Pediatric
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