The Activity Spectrum of Vif from Multiple HIV-1 Subtypes against

APOBEC3G, APOBEC3F, and APOBEC3H

Mawuena Binka,a Marcel Ooms,a Myeika Steward,a and Viviana Simona,b,c
Department of Microbiology,a The Global Health and Emerging Pathogens Institute,b and Division of Infectious Diseases, Department of Medicine,c Mount Sinai School of
Medicine, New York, New York, USA

The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees
in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the protea-
some. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3
proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and
A3H haplotype II (hapII). We show that most Vif alleles neutralize A3G and A3F efficiently but display differences with respect
to the inhibition of A3H hapII. The majority of non-subtype B Vif alleles tested presented some activity against A3H hapII, with
two subtype F Vif variants being highly effective in counteracting A3H hapII. The residues required for activity were mapped to
two residues in the amino-terminal region of Vif (positions 39F and 48H). Coimmunoprecipitations showed that these two
amino acids were necessary for association of Vif with A3H hapII. These findings suggest that the A3H hapII binding site in Vif is
distinct from the regions important for A3G and A3F recognition and that it requires specific amino acids at positions 39 and 48.
The differential Vif activity spectra, especially against A3H hapII, suggest adaptation to APOBEC3 repertoires representative of
different human ancestries. Phenotypic assessment of anti-APOBEC3 activity of Vif variants against several cytidine deaminases
will help reveal the requirement for successful replication in vivo and ultimately point to interventions targeting the Vif-
APOBEC3 interface.

A polipoprotein B mRNA-editing, catalytic polypeptide

(APOBEC3) cytidine deaminases act as intracellular inhib-
itors of HIV-1 replication (1, 8). The family comprises seven pro-
teins (APOBEC3A [A3A] to A3H), which differ in catalytic activ-
ity and susceptibility to HIV-1 Vif-mediated degradation (1, 8,
38). In the absence of a functional Vif, some APOBEC3 proteins
(e.g., A3G, A3F, and A3H haplotype II [hapII]) exert restriction
through editing- and nonediting-based mechanisms, thereby pre-
venting efficient viral spread in the next round of infection (1,
8, 13). HIV-1 isolates harbor a Vif allele that degrades A3G and
some other antiretroviral APOBEC3 proteins in a ubiquitin/
proteasome-dependent manner (1, 8, 38).
A3G and A3F contain two deaminase domains while the A3H
protein consists of only one catalytic domain. Moreover, A3H has
several haplotypes, some of which are highly active against HIV-1
(haplotypes II, V, and VII) (5, 9, 18, 19, 29, 43, 52). Interestingly,
the allele frequency of the active A3H hapII differs considerably
among human ethnicities, being high in African and low in Euro-
pean and Asian populations (29, 43). There remains some uncer-
tainty with respect to the Vif neutralization sensitivity of these
A3H protein variants (5, 10, 41, 52).
The global HIV-1 epidemic is mostly driven by non-subtype B
virus strains from group M (12), but our current knowledge on
functional determinants of Vif-APOBEC3 interactions is almost
exclusively based on the interactions of HIV-1 subtype B molecu-
lar clones (LAI, HXB2, and NL4-3) with the reference A3G. These
studies have generated a body of evidence indicating that the
amino-terminal region of Vif is important for binding to A3G and
A3F proteins (3, 16, 23, 24, 26, 34, 35, 37, 42, 44). Vif residues that
are important for selective association with A3G/A3F vary: Vif
residues 14 to 17 (DRMR) are important for interaction with A3F,
whereas residues 40 to 44 (YRHHY) are required for A3G binding
(26, 34, 35, 49). Other studies showed that Vif residues I9, K22,
E45, and N48 are necessary for A3G binding, whereas Q12 is
needed for A3F binding (37, 44). In addition, tryptophans at po-
sitions 11 and 79 and at positions 5, 21, 38, and 89 are important
for A3F and A3G binding, respectively (42). The YXXL (X stands
for any residue) motif spanning positions 69 to 72 and the WX
SLVK motif from positions 21 to 26 are necessary for the degra-
dation of both A3G and A3F (6, 32).
The C-terminal region of Vif contains the SLQYLA SOCS box
(residues 145 to 151), which is important for Vif binding to
elongin C in the E3 ubiquitin ligase complex (39, 50). This region
is highly conserved among HIV-1 subtypes, and mutations within
this domain disrupt the interaction between Vif and Elongin C,
thereby abrogating anti-APOBEC3 activity (20, 36). This region
also contains the zinc binding HCCH motif consisting of residues
H108, C114, C133, and H139, which are necessary for Vif binding
to Cullin 5, which mediates polyubiquitination of APOBEC3 pro-
teins, followed by their degradation via the 26S proteasome (21,
25, 31, 47, 48). To date, the three-dimensional structure of Vif has
not yet been solved by X ray or nuclear magnetic resonance
(NMR), but a conformational analysis of HXB2 Vif by hydrogen
exchange mass spectrometry suggested that the N-terminal Vif
region is more structured than the C-terminal portion of the Vif
protein (22). Taken together, our current understanding suggests
that the amino-terminal region of HIV-1 Vif allows APOBEC3-
Received 23 August 2011 Accepted 13 October 2011
Published ahead of print 19 October 2011
Address correspondence to Viviana Simon, viviana.simon@mssm.edu.
Supplemental material for this article may be found at http://jvi.asm.org/.
Copyright 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.06082-11

0022-538X/12/$12.00 Journal of Virology p. 49 59 jvi.asm.org 49

Binka et al.
specific interactions while the C-terminal region of Vif connects it
to the host cell degradation machinery.
Although Vif variants have been characterized genotypically
(2, 40, 45, 46), we know very little about the range of phenotypic
activity of non-subtype B Vif variants against the different
APOBEC3 proteins. Indeed, naturally occurring single residue
substitutions may affect the Vif/APOBEC3 phenotype, with full-
length subtype B Vif alleles displaying neutralization defects
against A3G, A3F, or both deaminases (27, 37). A recent study
compared the anti-A3G activity of Vif alleles derived from various
subtypes and recombinants (A, B, C, CRF01, and CRF02) and
found that subtype C Vif alleles displayed higher activity against
A3G (14). The correlates of the increased activity were mapped to
several residues in the amino-terminal region of Vif (14), but our
understanding of the requirements of diverse Vif proteins to
counteract multiple APOBEC3 proteins remains incomplete.
We hypothesized that Vif alleles, especially those obtained
from non-B subtype isolates that adapted to replication in indi-
viduals of different ancestries, may differ in their relative activities
against A3G, A3F, and A3H hapII. In this study, we show that
most Vif alleles from different HIV-1 subtypes have activity
against A3G and A3F. In contrast, only half of the Vif alleles tested
were partially or fully active against A3H hapII. Two distinct res-
idues in Vif (39F and 48H) were required for the efficient neutral-
ization of A3H hapII by Vif, suggesting that the single deaminase
A3H may interact with a nonlinear domain in Vif.
MATERIALS AND METHODS
Cloning. Twelve viral isolates and five molecular clones from different
subtypes were obtained through the NIH AIDS Research and Reference
Reagent Program (Table 1). Briefly, viral RNA was extracted from the viral
stocks using a Qiagen QIAamp Viral RNA Mini Kit, followed by reverse
transcription and amplification using primers located outside the Vif cod-
ing region. The PCR fragments were cloned into pSC-A (StrataClone PCR
cloning vector; Stratagene), and several clones were sequenced and man-
ually aligned to the published sequences. Clones with Vif sequences iden-
tical to the published sequences were used as templates for PCR amplifi-
cation with Vif primers specific for the 5= and 3= regions of each variant
and containing NotI and EcoRI restriction sites. PCR fragments were
digested with NotI/EcoRI, and the coding regions of the subcloned 17 Vif
variants were inserted in pCRV1 as described previously (37, 51). Site-
directed mutagenesis of subtype F1 and F2 Vifs was performed using
overlapping PCR as described previously (37). The mutated Vifs were
cloned into pCRV1 vector and confirmed by sequencing. Primer se-
quences are available upon request.
Amino-terminally FLAG-tagged A3G, A3F, and A3H hapII in PTR600
expression plasmids have been described previously (10, 28, 30). A
carboxyl-terminal triple hemagglutinin (HA) tag was added to A3G and
A3H hapII by overlapping PCR, followed by cloning into PTR600. An
untagged version of A3H hapII was cloned into PTR600. All DNA prep-
arations were sequenced to confirm the integrity of the APOBEC3 se-
quences. NCBI reference sequence numbers for the APOBEC3 used are
NM 021822.3 (A3G), NM 145298.5 (A3F), and FJ376614.1 (A3H hapII,
splice variant SV-183).
Cell culture. HEK 293T and TZM-bl cells were grown in Dulbeccos
modified Eagles medium (Invitrogen) supplemented with 100 U/ml pen-
icillin/streptomycin and 10% fetal bovine serum (FBS). TZM-bl cells were
provided by J. C. Kappes and X. Wu through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, National Insti-
tutes of Health, NIH Reagent program.
Infectivity assay. To determine the level of infectivity rescue achieved
by each Vif variant, viral stocks were produced in the presence and ab-
sence of APOBEC3. Briefly, 3.0 105 HEK 293T cells were cotransfected
50 jvi.asm.org
with 500 ng of pNL4-3Vif, 5 ng of Vif, and 50 ng of PTR600FLAG-A3G,
50 ng of PTR600FLAG-A3F, or 25 ng of PTR600FLAG-A3H expression
plasmids, using 4 g/ml polyethylenimine (PEI; Polysciences, Inc.).
The total amount of DNA transfected was 1,000 ng, with pcDNA3.1 serv-
ing as filler. Viral supernatants were collected at 44 to 48 h posttransfec-
tion, clarified by centrifugation, and stored at 80C. Forty-microliter
supernatants were used to infect 1 104 TZM-bl cells in black 96-well
plates. Infections were done in triplicate with viral supernatants from
three independent experiments for each APOBEC3 protein tested. Infec-
tivity of the virus particles in the TZM-bl cells was assessed at 44 to 48 h
postinfection using a Galacto-Star System for detecting -galactosidase
activity (Applied Biosystems), as described previously (10, 37).
Vif expression. A total of 3.0 105 HEK 293T cells were transfected
with 200 ng of Vif expression plasmids, 100 ng of PTR600-green fluores-
cent protein (GFP), and 700 ng of pcDNA (24-well plate; the total amount
of DNA transfected was 1,000 ng with 4 g/ml PEI). SDS lysis buffer (1%
SDS, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA) was added to
the transfected cells 48 h later, and the clarified lysates were analyzed by
Western blotting.
Degradation assay. A total of 3.0 105 HEK 293T cells in 24-well
plates were cotransfected with 500 ng of pNL4-3Vif, 100 ng of PTR600-
GFP, 20 ng of pCRV1-Vif, and 100 ng of PTR600FLAG-A3G, 100 ng of
PTR600FLAG-A3F, or 50 ng of PTR600FLAG-A3H hapII expression
plasmids (total amount of DNA transfected, 1,000 ng; filler DNA,
pcDNA3.1).
The degradation of tagged and untagged A3H hapII was compared by
cotransfecting HEK 293T cells as described above with the following ex-
pression plasmids: PTR600FLAG-A3H hapII (50 ng) and pCRV1-Vif (50
ng) or PTR600-A3H hapII (untagged; 100 ng) with pCRV1-Vif (100 ng).
The total amount of DNA transfected was 1,000 ng (filler DNA, pcDNA).
Transfections were performed with PEI (4 g/ml). At 48 h posttransfec-
tion cells were lysed (in lysis buffer consisting of 1% SDS, 50 mM Tris-
HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA), clarified by centrifugation, and
analyzed by Western blotting.
Western blotting. Transfected HEK 293T cell lysates were separated
on 10% bis-Tris 26-well gels (Invitrogen) and transferred by semidry
transfer onto polyvinylidene difluoride (PVDF) membranes (Thermo
Scientific). Membranes were blocked in 1% (wt/vol) milk solution for 1 h
and incubated in primary antibody overnight. HIV-1 Vif was detected
with a rabbit anti-Vif primary antibody (NIH catalog number 2221) at
1:1,000 dilution and with goat anti-rabbit secondary antibody (1:5,000;
Sigma). FLAG-tagged APOBEC3 proteins were detected with a mouse
anti-FLAG primary antibody (1:5,000; Sigma), GFP was detected
with rabbit anti-GFP primary antibody (1:2,000; Santa Cruz), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected with
a mouse anti-GAPDH primary antibody (1:2,000; Santa Cruz). Goat anti-
rabbit or goat anti-mouse secondary antibody (both, Sigma) was at a
1:5,000 dilution. Membranes were washed in wash buffer (0.1% Tween in
phosphate-buffered saline [PBS]) and incubated in secondary antibody
for 2 h. Membranes were developed with SuperSignal West Pico or Femto
substrate (Thermo Scientific) in an LAS-3000 imaging system (FujiFilm).
Only nonsaturated signals were acquired for further analysis with Image-
Gauge, version 4.0, software.
Vif-A3H coimmunoprecipitation. A total of 7 105 HEK 293T cells
were transfected with pCRV1-Vif expression plasmids (200 ng),
PTR600HA-A3H hapII (800 ng), PTR600HA-A3G (800 ng), or PTR600-
GFP (800 ng) using Fugene 6 according to the manufacturers instruc-
tions. At 24 h after transfection, clasto-lactacystin -lactone (10 M;
Sigma) was added to the transfection to prevent potential Vif-mediated
A3H degradation. At 48 h after transfection, cells were washed with PBS
and lysed on ice for 15 min in lysis buffer (1% Triton X-100 in PBS
supplemented with EDTA-free protease inhibitor cocktail [Roche]). Pro-
teins were clarified by centrifugation at 4C (8,200 g for 10 min at 4C)
and incubated with anti-HA-coated beads (Easyview; Sigma) for 1 h at
4C. Beads were extensively washed with lysis buffer supplemented with
Journal of Virology
 Activity of Vif from HIV-1 Subtypes against APOBEC3

TABLE 1 Description of the Vif alleles used in this study Statistical analysis. GraphPad Prism, version 5, software was used to
Vif name Strain name Country of origin perform all statistical tests (minimum, maximum, mean, t tests, and
Spearman rank correlation). P values are two sided, and values of 0.05
A1 93RW037 Rwanda were considered to be significant.
A2 92RW008 Rwanda
B-NL4-3 pNL4-3 USA RESULTS
B-LAI pLAI.2 USA Genotype and expression of selected Vif alleles. Inspection of
B1 92TH026 Thailand
1,500 Vif alleles deposited at the Los Alamos HIV sequence da-
B2 AD.MDR01 USA
B3 93TH305 Thailand
tabase revealed considerable variation in Vif sequences. Some
C1 92ZW101 Zimbabwe subtype-specific motifs such as 8L (subtype C), 45DCXH48 (sub-
C2 92ZW106 Zimbabwe type D), and 91QRK93 (subtype AE) were also identified. We
C3 pMJ4 Botswana selected 12 viral isolates from patients representing seven HIV-1
D1 p94UG114.1.6 Uganda subtypes and five replication-competent full-length molecular
D2 94KE102 Kenya clones from three different subtypes. The subtype-specific motifs
AE1 p90CF402.1.8 Central African Republic listed above were present in the selected Vif alleles. The patients
F1 93BR029 Brazil from whom the viruses were originally isolated lived in various
F2 93BR019 Brazil countries around the world, like Brazil, Rwanda, Zimbabwe, and
F3 93BR020 Brazil
Thailand (Table 1). Analysis of the phylogenic relationships of the
G1 HIV-1 G3 Nigeria
selected Vif alleles and their corresponding consensus sequences
confirmed their subtypes (Fig. 1A).
At the protein sequence level, the 17 Vif alleles displayed, on
75 mM NaCl. Bound proteins were eluted by boiling the beads in sample average, 15% diversity relative to HIV subtype B molecular clone
loading buffer. Cell lysates and bound proteins were analyzed by Western LAI (maximum, 19%; minimum, 7%). The greatest number of
blotting.
changes were observed for Vif G1 (37/192 residues), while Vif B3
A3H polyclonal serum. Two rabbits were immunized three times with
the A3H peptide Gln-Phe-Asn-Asn-Lys-Arg-Arg-Leu-Arg-Arg-Pro-Tyr- (29/192 residues) was most similar to LAI Vif. Inspection of the
Tyr-Pro-Arg (A3H residues 12 to 26). Custom antibody production was N-terminal region revealed polymorphic positions both within
performed by Pocono Rabbit Farm and Laboratory, Inc. Rabbit poly- and outside the domains known to be relevant for APOBEC3-
clonal serum was used at a 1:2,000 dilution to detect transfected untagged specific interactions while the HCCH Cullin 5-binding motif and
and tagged A3H. the SLQYLA Elongin C-binding motif were conserved in all 17 Vif

FIG 1 A panel of 17 Vif alleles from different subtypes. (A) Phylogenetic relationships of subtype consensus sequences and selected Vif alleles from different
subtypes were inferred using the neighbor-joining method. Bootstrap values of 70% or higher are shown next to the branches (1,000 replicates). Distances were
computed using the maximum composite likelihood method. All positions containing gaps and missing data were eliminated from the data set (complete
deletion option). There were a total of 568 positions in the final data set. Phylogenetic analyses were conducted in MEGA4. (B) Vif variants from different subtypes are
expressed to comparable levels. pCRV1 Vif expression plasmids were transfected with GFP expression plasmid into HEK 293T cells. Lysates were separated by SDS-PAGE
at 48 h posttransfection, transferred to PVDF membranes, and probed with antibodies against Vif, GFP, and GAPDH. (C) Relative Vif expression for each Vif variant is
shown. Nonsaturated signals were acquired, background was subtracted, and values were normalized to GFP expression levels. NL4-3 Vif expression was set to 1. The
average and standard deviations of three independent experiments are shown. Error bars represent standard deviations. denotes anti.

January 2012 Volume 86 Number 1 jvi.asm.org 51

Binka et al.

FIG 2 Activity of Vif alleles against A3G and A3F. (A) Anti-A3G activities of the different Vif variants. Infectivity of pNL4-3Vif cotransfected with Vif alleles
and A3G was assessed at 48 h posttransfection on TZM-bl reporter cells. Data shown are representative of three independent experiments. Error bars represent
standard deviations. Unpaired t tests were computed to determine whether differences between LAI Vif infectivity and a given Vif variant reached significance (,
P 0.05; , P 0.01; , P 0.001 [Prism software]). (B) Degradation of A3G by the different Vif alleles. Transfected lysates were separated by SDS-PAGE
at 48 h posttransfection, transferred to PVDF membranes, and probed with antibodies against FLAG, Vif, and GFP. One representative Western blot is shown.
(C) Anti-A3F activities of the different Vif variants. Infectivity of pNL4-3Vif cotransfected with Vif alleles and A3G was assessed at 48 h posttransfection on
TZM-bl reporter cells. Data shown are representative of three independent experiments. Error bars represent standard deviations. Unpaired t tests were
computed to determine whether differences between LAI Vif infectivity and a given Vif variant reached significance (, P 0.05; , P 0.01; , P 0.001
[Prism software]). (D) Degradation of A3F by the different Vif alleles. Transfected lysates were separated by SDS-PAGE at 48 h posttransfection, transferred to
PVDF membranes, and probed with antibodies against FLAG, Vif, and GFP. Representative Western blots are shown.

alleles (see Fig. S1 in the supplemental material for Vif sequence
alignments).
The majority of Vif variants were similarly expressed upon
transfection in 293T cells (Fig. 1B and C), with the exception of the
subtype C Vif C1, which consistently displayed a 2-fold decrease in
signal intensity (Fig. 1C). This difference may reflect lower Vif
protein expression or reduced antibody recognition by the poly-
clonal anti-Vif rabbit serum. All 17 Vif variants encode 192 resi-
dues, but some Vif bands migrated differently during gel electro-
phoresis, likely reflecting variations in protein charges (Fig. 1B,
compare Vif F1 to Vif F3 or Vif G1).
Anti-APOBEC3 phenotypes of Vif alleles. The different Vif
proteins were analyzed for rescue of infectivity of NL4-3 with a
deletion of Vif (pNL4-3Vif) in the presence of A3G, A3F, and
A3H hapII. In addition, APOBEC3 degradation by the Vif panel
was assessed by cotransfecting the Vifs and APOBEC3, followed
by Western blot analysis. Titrations of Vif expression plasmids (5
ng, 100 ng, and 500 ng) and APOBEC3 plasmids (25 ng, 50 ng, and
100 ng) in the presence of NL4-3Vif were performed to select
assay conditions and plasmid ratios that allowed optimal discrim-
ination of both an increase and a decrease in rescue of infectivity
(data not shown). Five nanograms of Vif and 50 ng of A3G or 50
ng of A3F were used in the experiments comparing the anti-
A3G/A3F activities of different Vif variants. In addition, simi-
lar experiments were performed with Vif-deleted HIV-1 mo-
lecular clones pLAI, pMJ4 (subtype C, Vif C3), and p94UG114
(subtype D, Vif D1) to exclude potential isolate and subtype
incompatibilities (data not shown). Of note, Capsid p24 pro-
duction upon transfection was comparable for all viral stocks
(data not shown).
Most HIV-1 Vif alleles counteract A3G and A3F restriction.
The infectivity of viral stocks was quantified 48 h posttransfection
using TZM-bl reporter cells as described previously (10, 28, 37).
An empty pCRV1 plasmid and the NL4-3 Vif mutant C133S,
which abrogates cullin 5 binding (C133S), were used as negative
controls. Infectivity of NL4-3Vif produced in the absence of
both APOBEC3 and Vif was set to 100% relative infectivity (Fig.
2A and C; see also Fig. 3A). We chose the widely used molecular
clone LAI Vif variant as a reference for comparison to other Vifs
(Fig. 1A), and the differences relative to LAI Vif were calculated
using a t test (Prism GraphPad).
In the absence of Vif or in the presence of the Vif C133S mu-
tant, A3G strongly reduced infectivity to 1%. The reference LAI
Vif rescued infectivity from A3G restriction to 60% 5%, and the
majority of Vif alleles tested were similarly or more active than LAI
Vif (Fig. 2A).
Four Vif variants (NL4-3, B2, C1, and G1) displayed low to
moderate activities against A3G (relative infectivity, 35% 4.5%,
36% 2.1%, 10.3% 3.5%, and 35% 2.5%, respectively), and
only one Vif allele (B3) was fully defective against A3G (relative
infectivity, 2.7% 1.5%). Interestingly, Vif C1, C2, and C3 were
expressed to lower levels than the other Vifs, and correcting infec-
tivity data with the Vif expression levels shown in Fig. 2A and B
demonstrated that all subtype C Vifs were relatively more active
against A3G.
The ability of the Vifs to degrade the APOBEC3s was deter-
mined by transfecting HEK 293T cells with 20 ng of the different
Vif expression plasmids and 100 ng of A3G (Fig. 2B) or A3F (Fig.
2D). The rescue of infectivity correlated with the ability to degrade
A3G in the producer cell, with the exception of Vif C1, which

52 jvi.asm.org Journal of Virology

 Activity of Vif from HIV-1 Subtypes against APOBEC3

FIG 3 Activity of Vif alleles against A3H hapII. (A) Anti-A3H hapII activities of the different Vif variants. Infectivity of pNL4-3Vif cotransfected with the Vif
alleles and A3H hapII was assessed at 48 h posttransfection on TZM-bl reporter cells. Data shown are representative of three independent experiments. Error bars
represent standard deviations. Unpaired t tests were computed to determine whether differences between LAI Vif infectivity and a given Vif variant reached
significance (, P 0.05; , P 0.01; , P 0.001 [Prism software]). No Vif/No A3H, 100% relative infectivity. (B) Degradation of A3H hapII by the different
Vif alleles. Transfected lysates were separated by SDS-PAGE at 48 h posttransfection, transferred to PVDF membranes, and probed with antibodies against FLAG,
Vif, and GFP. Representative Western blots of one out of three experiments are shown. No Vif/A3H, 100% A3H remaining. (C) A3H hapII degradation efficiency
of different Vif variants. A3H hapII signals were quantified and the no-Vif control is set at 100%. Error bars represent standard deviations of three independent
A3H hapII degradation assays. (D) Correlation between Vif-mediated rescue of viral infectivity in the presence of A3H hapII and Vif-mediated degradation. Gray
symbols identify the controls (as described for A and B). (E) The pattern of A3H hapII-mediated degradation by Vif is independent of the FLAG tag. Selected
active and nonactive Vif variants were transfected with FLAG-tagged and untagged A3H hapII expression plasmids. Transfected lysates were separated by
SDS-PAGE at 48 h posttransfection and transferred to PVDF membranes. Proteins were detected with polyclonal rabbit serum directed against A3H or Vif. GFP
was detected with a monoclonal antibody.

degraded A3G efficiently but failed to rescue viral infectivity to the
same degree.
A3F is less potent than A3G in restricting NL4-3Vif comple-
mented with an empty control plasmid or with the mutant Vif
C133S (no-Vif and Vif C133S, 10% compared to 1% with A3G).
LAI Vif rescued infectivity in the presence of A3F to 64% 5%,
with all Vif alleles displaying activity against A3F. Vif variants AE1,
F2, and F3 were more active than LAI Vif (relative infectivity,
73% 8.1%, 85% 8.5%, and 71% 6.6%, respectively). Vif
C1, which displayed low activity against A3G, showed average
activity against A3F (relative infectivity, 50% 2.0%). Interest-
ingly, Vif B3, which was not active against A3G, displayed some
activity against A3F (relative infectivity, 26% 3.1%) although its
ability to degrade A3F was modest (Fig. 2D). Overall, most Vifs
were able to degrade and counteract A3G and A3F restriction.
Most non-subtype B HIV-1 Vif alleles counteract A3H hapII
restriction. More than eight naturally occurring A3H haplotypes
have been described, with A3H hapII having potent anti-HIV ac-
tivity (10, 29, 43). There remains some uncertainty about the ex-
tent to which A3H is partially or completely resistant to Vif neu-
tralization (5, 10, 29, 41, 43, 52). We therefore tested whether the
different Vif alleles have activity against A3H hapII (Fig. 3A).
Based on initial optimization experiments (data not shown), we
used less APOBEC3 expression plasmid for the experiments with

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Binka et al.
FIG 4 Comparison of the anti-APOBEC3 activities of different Vif alleles. A heat map representation illustrates the spectrum of infectivity obtained with each
Vif variant in the presence of A3G, A3F, and A3H hapII. Normalized infectivity values are plotted. The controls for each deaminase are shown on the right side
of the panel: the level of suppression achieved (%) in the absence of Vif is depicted (red), and the infectivity (%) in the absence of APOBEC3 is set to 100 (green).
Relative infectivity values are the average of three independent experiments shown in Fig. 2 and 3. Max denotes maximum.
A3H hapII (25 ng of A3H hapII compared to 50 ng of A3G or
A3F). Under these experimental conditions, A3H hapII reduces
infectivity to 10% 1.0% in the absence of Vif or with the mutant
Vif C133S.
Interestingly, 9 out of 17 Vif variants were able to rescue infec-
tivity in the presence of A3H hapII. Of the active Vifs, Vif F1 and
F3 were, by far, the most efficient at counteracting A3H hapII (F1,
80% 7.5%; F3, 77% 8.0%) (Fig. 3A), followed by LAI, two
subtype C Vif variants (C1 and C3), and one subtype A Vif variant
(A1). Of note, Vif C1, which had low activity against A3G and
modest activity against A3F, was active against A3H hapII (35%
2.0%). Three out of four subtype B variants failed to counteract
A3H hapII restriction, with LAI Vif being the sole exception. LAI
Vif rescued viral infectivity in the presence of A3H hapII to 43%
6.1%, whereas NL4-3 Vif was defective in rescuing A3H-mediated
restriction (11% 2.6%). This observation is in good agreement
with reports showing that Vif from LAI but not NL4-3 is active
against A3H hapII (18, 19).
The ability of Vif variants to degrade the A3H hapII was deter-
mined by transfecting HEK 293T cells with 20 ng of the different
Vif expression plasmids and 50 ng of A3H (Fig. 3B). Overall, A3H
hapII was more resistant to degradation than A3G and A3F (Fig.
3B, compare the no-Vif and Vif C133S control levels to the level of
A3H in the presence of other Vifs). The Vif variants LAI, F1, and
F2 degraded A3H hapII efficiently (Fig. 3C, e.g., only 20% of the
no Vif A3H hapII control remaining), whereas the other Vif
variants were partially or completely defective with respect to deg-
radation in the producer cell (Fig. 3B and C). The degree of Vif-
mediated rescue of viral infectivity in the presence of A3H hapII
correlated well with the efficiency to degrade A3H hapII (Fig. 3D).
Adding epitope tags to A3H hapII has been reported to influ-
ence Vif-mediated A3H hapII degradation (18). To test whether
the N-terminal FLAG tag to A3H hapII affects its susceptibility to
Vif, we analyzed 10 Vif alleles with N-terminal FLAG tags and
untagged A3H hapII for degradation. We transfected the selected
Vif variants and the two A3H expression plasmids into HEK 293T
cells and used polyclonal rabbit serum raised against an A3H pep-
tide to detect both A3H hapII variants in the cell lysates by West-
ern blotting (Fig. 3E). The A3H hapII degradation patterns were
similar for the N-terminally tagged and untagged A3H hapII pro-
teins (Fig. 3E), indicating that the N-terminal FLAG tag did not
affect its sensitivity to Vif degradation.
Taken together, in contrast to the efficient activity of most of
the Vifs against A3G and A3F, Vifs from different subtypes dis-
played considerable differences in counteracting A3H hapII re-
striction.
54 jvi.asm.org
The spectrum of anti-APOBEC3 activities varies between Vif
variants. Since multiple APOBEC3 proteins are expressed in
HIV-1 susceptible cells, we were interested in a comprehensive
assessment of Vif-mediated anti-APOBEC3 activities. Figure 4
provides a heat map representation of the anti-APOBEC3 spec-
trum of each Vif allele (complete rescue indicated in green and
maximum restriction shown in red). Vif variants F1 and F3
showed high activity against all three deaminases tested (broad-
spectrum anti-APOBEC3 activity), while Vif B3 displayed low ac-
tivity against all three APOBEC3s. Interestingly, we also noted
A3-specific Vif defects in which one (Vif C1 and Vif F2) or two
(Vif C2) specific APOBEC3s were not neutralized. Overall, the
patterns of rescue from A3G and A3F were similar among Vifs but
did not correlate with the activities of these Vifs against A3H ha-
pII. This indicates that residues in Vif that interact with A3G and
A3F are likely different from those that interact with A3H hapII.
Identification of the Vif residues required for A3H hapII deg-
radation. Subtype F Vif variants were efficient in counteracting
A3G and A3F but differed in their abilities to block A3H hapII
(Fig. 4). This apparent difference between F1 and F2 with respect
to A3H hapII was used to accurately determine the Vif require-
ments for interacting with A3H hapII. We focused on the
N-terminal part of Vif because this region has been previously
shown to interact with A3G and A3F. Since Vif F1 and Vif F2
differ at only five residues in the N-terminal region (positions 39,
48, 61, 62, and 63) (Fig. 5A), we generated a series of mutants in
which residue 39, residue 48, or residues 61 to 63 were exchanged
between the two Vifs (Fig. 5A). We tested the infectivity of NL4-
3Vif with these Vif mutants in the presence and absence of A3H
hapII (Fig. 5B). Infectivity data showed that two residues, 39F and
48H, are responsible for the difference between Vif F1 and Vif F2
and that both are required for efficient anti-A3H hapII activity.
Replacing either the phenylalanine (F) at position 39 with a serine
(39S) or the histidine (H) at position 48 with an asparagine (48N)
rendered Vif F1 inactive against A3H hapII, while the introduc-
tion of both residues (F39 and H48) was required to render Vif F2
active against A3H hapII (Fig. 5B). The ability of these mutants to
degrade A3H hapII was also assessed (Fig. 5C) and showed that the
ability to rescue infectivity correlated with A3H hapII degradation
(Fig. 5B and C). Interestingly, mutations at these two positions
affected only the activity against A3H hapII, whereas the ability to
counteract A3G and A3F was unaffected (data not shown).
Before Vif can mediate the degradation of APOBEC3 by the
proteasome, both proteins must first physically interact. We spec-
ulated, therefore, that the Vif proteins that failed to counteract
A3H hapII might be inefficiently interacting with A3H hapII. We
Journal of Virology
 Activity of Vif from HIV-1 Subtypes against APOBEC3

FIG 5 Identification of the residues important for activity of subtype F Vif variants against A3H hapII. (A) A schematic of the Vif N-terminal mutants generated
to investigate activity against A3H hapII. Vif F1 and Vif F2 differ at positions 39, 48, and 61 to 63. (B) Infectivities of pNL4-3Vif cotransfected with Vif F1, Vif
F2, and Vif mutants M1 to M8 and A3H hapII were assessed at 48 h posttransfection on TZM-bl reporter cells. Data shown are representative of three
independent experiments. Error bars represent standard deviations of triplicate experiments. (C) Western blot depicting A3H hapII degradation in the presence
of Vif F1, Vif F2, and Vif mutants M1 to M8. Transfected lysates were separated by SDS-PAGE at 48 h posttransfection, transferred to PVDF membranes, and
probed with antibodies against FLAG, Vif, and GFP. Data shown are representative of three independent experiments. (D) Coimmunoprecipitation (Co-IP) of
subtype F Vif variants with A3H hapII and A3G. Subtype F Vif F1, Vif F2, or empty control plasmid was cotransfected with HA-tagged A3H hapII, HA-tagged
A3G, or control plasmid. APOBEC3 degradation was prevented by the addition of the proteasome inhibitor clasto-lactacystin . Cleared lysates were incubated
with anti-HA-coated beads and washed vigorously. Proteins were analyzed by Western blotting. (E) Coimmunoprecipitation of subtype F Vif variants with A3H
hapII. Subtype F Vif F1, Vif F2, and Vif mutants M4 and M3 were cotransfected with HA-tagged A3H hapII. A3H hapII degradation was prevented by the addition
of the proteasome inhibitor clasto-lactacystin . Cleared lysates were incubated with anti-HA-coated beads and washed vigorously. Proteins were analyzed by
Western blotting.

performed coimmunoprecipitation experiments by transfecting
Vif F1 and Vif F2 with A3H hapII or A3G in HEK 293T cells. The
proteasome inhibitor clasto-lactacystin -lactone was added to
the medium 24 h before lysis to prevent APOBEC3 degradation.
Vif F1 was more efficient in precipitating A3H hapII than Vif F2,
whereas both Vif F1 and F2 associated efficiently with A3G (Fig.
5D). In addition, we established that the two residues were neces-
sary and sufficient to determine the ability of Vif F variants to
associate with A3H hapII by cotransfected Vifs F1 and F2 as well as
mutant Vifs M4 and M3 with A3H hapII in HEK 293T cells. Vif F1
determine the association between subtype F Vifs and A3H hapII
(Fig. 5E). These results correlated well with the observed infectiv-
ity and degradation phenotypes (Fig. 5B and C).
Alanine scanning of residues 38 to 49 in the amino-terminal
region of subtype F Vif. In addition to the polymorphic nature of
the amino acids at position 39 and 48, changes within the region
between these two residues may affect the interaction between Vif
and APOBEC3. Indeed, several motifs in this region have been
described to affect subtype B Vif function against A3G (e.g.,
40YRHHY44, 45E, and 48N) (34, 37). We individually replaced all

and mutant M3 (with residues 39F and 48H) immunoprecipitated amino acids between positions 38 and 49 in Vif F1 with alanines
with A3H hapII, while defective Vifs F2 and mutant M4 (with 39S and analyzed their effects on rescue of infectivity in the presence of
and 48N) both failed to associate with A3H hapII. This indicates A3G, A3F, and A3H hapII. The alanine scanning of this Vif F1
that a phenylalanine at position 39 and a histidine at position 48 region showed that distinct positions had differential effects on

January 2012 Volume 86 Number 1 jvi.asm.org 55

Binka et al.

FIG 6 Characterization of the Vif/APOBEC3 interface that allows degradation of A3H hapII in the Vif F1 context. The anti-APOBEC3 activities of the 12 different
alanine mutants in Vif F1 (alanine scanning mutagenesis of the region between residues 38 and 49) are shown. Infectivity of pNL4-3Vif cotransfected with the Vif
mutants and A3G (A), A3F (B), and A3H hapII (C) was assessed 48 h posttransfection on TZM-bl reporter cells. Data shown are representative of three independent
experiments. Error bars represent standard deviations. Western blots showing A3G, A3F, and A3H hapII degradation by the mutant F1 Vifs are directly under the
respective infectivity values. (D) Transfected Vif F1 mutants are expressed to comparable levels. Representative Western blots are shown for Vif expression and the GFP
transfection control. (E) Heat map summarizing the anti-A3G, -A3F, and -A3H hapII phenotypes associated with each Vif F1 alanine mutant.

activity against A3G, A3F, and A3H hapII (Fig. 6). Alanines at
position 38, 48, and 49 rendered Vif F1 defective against all three
APOBEC3s (Fig. 6A, B, C, and E) but did not affect Vif expression
(Fig. 6D). None of the other mutations reduced neutralization of
A3F, while other mutations showed APOBEC3-specific effects:
40A completely abrogated anti-A3G and anti-A3H hapII activity,
and several substitutions in this region resulted in a specific loss of
function against A3H hapII (41A, 40A, and 45A) (Fig. 6C). Posi-
tion 39, one of the two positions that determined the difference
between Vif F1 and F2 with regard to A3H degradation, was more
tolerant to being mutated to an alanine (from an aromatic phe-
nylalanine) than to the nucleophilic serine. Western blot analysis
showed that the efficiency in rescue of infectivity correlates with
the level of APOBEC3 degradation by the respective Vifs. Taking
these results together, alanine scanning mutagenesis of the region
between residues 39 and 48 in subtype F Vif suggests that the
residues necessary for A3H hapII recognition are distinct from
those required for Vif-A3G or Vif-A3F interactions.
Next, we analyzed the sequences between positions 38 and 49
of the initial subtype set of Vifs to understand why only some Vifs
counteract A3H hapII. Figure 7 shows an alignment of the protein
region of interest (positions 38 to 49) of the 17 Vif proteins tested
and their respective activities against A3H hapII. Nearly all Vifs
that efficiently counteracted A3H hapII carried the 39F and 48H
combination, whereas most of the inactive Vifs carried alternative
pairs. Only a few exceptions existed: Vif B2 contains the FH pair
but lacks anti-A3H activity, which could be caused by the unique
glycine (G) at position 41 (41G, all other active Vifs carry 41R).
This possibility is further supported by the alanine scanning re-
sults, which showed the importance of position 41 for anti-A3H
function (Fig. 6C). Vif D2, which carries an LH pair instead of the
FH pair, displayed low activity against A3H hapII. The leucine (L)
at position 39 shares the same hydrophobic and nonpolar prop-
erties as the phenylalanine (F), indicating that some variation at
position 39 is tolerated with amino acids of similar properties, like
39A (Fig. 6C).
In summary, a 39F-48H or a 39L-48H pair is required for ac-
tivity against A3H hapII but not for neutralization of A3G and
A3F. In addition, amino acid changes at critical residues between
these positions (e.g., 40, 41, and 45) may abrogate anti-A3H hapII
function in the presence of the FH/LH pairs.
DISCUSSION
At least five cytidine deaminases of the APOBEC3 family restrict
retroviruses if left unimpeded by Vif (1, 4, 8, 11, 38). Our knowl-
edge of the anti-APOBEC3 spectra of diverse non-B subtype Vif
variants is limited to A3G (14).
In this study, we assessed whether Vifs from different HIV-1
subtype strains are able to counteract specific APOBEC3 proteins
(A3G, A3F, and A3H hapII) with comparable efficiencies. We

56 jvi.asm.org Journal of Virology


FIG 7 Correlation between Vif genotype and activity against A3H hapII. The
amino acid alignment of the 17 Vifs shows the region between residues 38 to 49
in the amino-terminal portion of Vif. Vif F1 serves as reference in this align-
ment. The Vifs are ranked in descending order of anti-A3H hapII activity (Fig.
4). Nine Vif variants (F1 to G1) rescued viral infectivity in the presence of A3H
hapII to 20% or more. These infectivity values were significantly higher (P
0.05, t test) from the no-Vif or the Vif C133S controls. The level of rescue is
indicated as follows: , high, with infectivity 50% of the no-A3H con-
trol; , moderate, with 50 to 30% of the no-A3H control; and , low, with
20 to 30% of the no-A3H control. Infectivity values of 20% of the no-A3H
control were defined as lack of neutralization activity (). The amino acid
pairs at locations 39 and 48 are grouped as FH, ZH, and XN. Z denotes any
residue except F, and X denotes any residue.
found that several non-B subtype Vif alleles were more efficient in
neutralizing A3G, A3F, and A3H hapII than the commonly used
subtype B Vifs. Indeed, most Vif alleles tested in this study against
A3G and A3F were at least as active as the Vif obtained from the
subtype B LAI molecular clone (Fig. 2 and 4). Moreover, the ma-
jority of non-B subtypes were able to neutralize A3H hapII (Fig. 3
and 7). The subtype F Vif variants F1 and F3 were especially effi-
cient in neutralizing A3H hapII (70% of the no-A3H control),
while a second group of Vifs (Vif A1, C1, C3, and LAI) displayed
moderate activity against A3H hapII (e.g., 30 to 50% infectivity
of the no-A3H control). Two residues, 39F and 48H, were re-
quired for this activity (Fig. 5). Alanine scanning mutagenesis re-
vealed that changes to other residues between positions 39 and 48
may affect the anti-A3H hapII function even in the presence of the
required 39F-48H pair (Fig. 6). The observed variation in anti-
A3H hapII activity of the Vifs tested in this study, however, was
largely explained by the presence/absence of this specific amino
acid pair (Fig. 7). For example, the difference in anti-A3H activities
between the LAI and NL4-3 Vifs is likely due to the substitution at
position 48, but the additional differences between NL4-3 and LAI
Vifs at positions 47 and 50 may also affect anti-A3H activity (Fig. 7). A
recent analysis of HIV-1 Vif sequence variation in an Argentinean
pediatric cohort (7) showed that approximately 70% of subtype F
strains encoded FH at positions 39/48, while 30% carried ZH or XN
combinations (where Z indicates any amino acid other than F, and X
January 2012 Volume 86 Number 1
Activity of Vif from HIV-1 Subtypes against APOBEC3
denotes any residue), suggesting that the majority of subtype F strains
likely counteract A3H hapII.
Our findings suggest that HIV-1 Vif variants display a spec-
trum of neutralization phenotypes, which cannot be easily pre-
dicted from the Vif genotype. Some Vifs failed to neutralize A3G
but rescued infectivity in the presence of A3F or A3H hap II (e.g.,
Vif C1), while others displayed activity against A3G and A3F but
not A3H hapII. We propose that the sum of these activities likely
determines the degree to which APOBEC3 restriction is overcome
in the context of given individuals APOBEC3 composition. More
comprehensive data sets relating Vif genotype to Vif phenotype
are needed to develop algorithms that allow prediction of anti-
APOBEC3 activities.
The structure-function experiments performed in this study
imply that the Vif interface recognizing A3H hapII is distinct from
the A3G or A3F interface. Multiple distant residues are involved,
suggesting a nonlinear recognition domain. We speculate that the
Vif alleles lacking activity against A3H hapII were originally ob-
tained from individuals who did not express active A3H variants
and, thus, did not require A3H neutralization. Interestingly, the
anti-APOBEC3 activity of LAI Vif was more potent as well as
broader than that of NL4-3 Vif; the LAI Vif not only neutralized
A3G and A3F with higher efficiency but also was the only subtype
B Vif that displayed activity against A3H hapII. Endogenous A3H
expression levels are believed to be modest, but the differential
neutralization spectra of several Vif variants of different subtypes
support the role of A3H as a restriction factor in vivo.
Some limitations of our study need to be mentioned. First, we
cannot draw generalized conclusions about subtype-specific ac-
tivities since the sample sizes are not sufficiently representative.
Moreover, the Vif alleles used in this study were cloned from
viral isolates obtained by coculture. Future studies on the anti-
APOBEC3 function should use Vif variants directly obtained from
the plasma of patients in order to obtain information on the Vif
activity spectrum without bias introduced by passaging the viral
stock in cells from a different donor. Moreover, experimental ap-
proaches using overexpression assays are frequently used to probe
for HIV-APOBEC3 interactions, but specific assays using primary
T lymphocytes encoding specific APOBEC3 haplotypes are
needed to further validate our observations at more physiological
APOBEC3 expression levels.
The APOBEC3 locus is polymorphic, with several haplotypes
for each of these deaminases (1). Proviruses with footprints of past
cytidine deamination are found in many infected patients inde-
pendently of the HIV-1 subtype (1, 15, 17, 33), suggesting that
variation in the spectra of the anti-APOBEC3 activities of HIV-1
Vif and/or antiretroviral activity of Vif-resistant APOBEC3 pro-
teins is important in vivo. HIV-1-infected patients with certain
A3H haplotypes displayed fewer G-to-A mutations and lower viral
loads (9), and it is attractive to speculate that Vif variants that
counteract multiple APOBEC3 proteins decrease the complexity
of the viral quasi-species as well as the pathogenicity of HIV-1 in a
given infected individual. A previous study found that Vif variants
from a patient harboring the active A3H hapII were active against
this variant while Vif variants from patients with A3H hapI failed
to degrade A3H hapII (19). This observation, together with our
findings, suggests a more complex picture in which Vif may adapt
to the APOBEC3 haplotype repertoire of the infected host. Future
studies looking for clinical associations between APOBEC3 and
jvi.asm.org 57
Binka et al.
HIV/AIDS disease progression will have to focus on multiple
APOBEC3 deaminases in conjunction with the Vif phenotype.
ACKNOWLEDGMENTS
We thank all the members of the Simon laboratory for helpful discussions.
This research was supported by National Institutes of Health grants
AI089246, AI90935, and AI064001.
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