Adaptation of HIV-1 to Its Human Host

Louise V. Wain,1 Elizabeth Bailes,1 Frederic Bibollet-Ruche,2 Julie M. Decker,2 Brandon F. Keele,2
Fran Van Heuverswyn,3 Yingying Li,2 Jun Takehisa,2 Eitel Mpoudi Ngole,4 George M. Shaw,2
Martine Peeters,3 Beatrice H. Hahn,2 and Paul M. Sharp1
1
Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom; 2Departments of Medicine
and Microbiology, University of Alabama at Birmingham; 3Laboratoire Retrovirus, Institut de Recherche pour le Développement and
Department of International Health, University of Montpellier, 34394 Montpellier cedex 9, France; and 4Projet Prevention du Sida au
Cameroun (PRESICA), Yaounde, Cameroun

Human immunodeficiency virus type 1 (HIV-1) originated from three independent cross-species transmissions of simian
immunodeficiency virus (SIVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving
rise to pandemic (group M) and non-pandemic (groups N and O) clades of HIV-1. To identify host-specific adaptations
in HIV-1 we compared the inferred ancestral sequences of HIV-1 groups M, N and O to 12 full length genome sequences
of SIVcpzPtt and four of the outlying but closely related SIVcpzPts (from P. t. schweinfurthii). This analysis revealed
a single site that was completely conserved among SIVcpzPtt strains but different (due to the same change) in all three
groups of HIV-1. This site, Gag-30, lies within p17, the gag-encoded matrix protein. It is Met in SIVcpzPtt, underwent
a conservative replacement by Leu in one lineage of SIVcpzPts but changed radically to Arg on all three lineages leading
to HIV-1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages
of HIV-1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV-1 was
passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we
used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and
tested their replication in both human and chimpanzee CD4þ T lymphocytes. Remarkably, viruses encoding
Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human
T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the
emergence of HIV-1 and identify the viral matrix protein as a modulator of viral fitness following transmission to the new
human host.

Introduction of the world. Group M strains have been diversifying since

around 1930 (Korber et al. 2000), and are classified into
Human immunodeficiency viruses types 1 and 2 (HIV-1
numerous subtypes. The protein sequences encoded by
and HIV-2) are the cause of HIV-AIDS. HIV-1 and HIV-2 are
contemporary strains of HIV-1 group M differ substantially
classified in the genus Lentivirus of the family Retroviridae.
from those of their closest known SIVcpz relatives (e.g., by
Related lentiviruses have been found infecting more than
14-35% in the major polyproteins Gag, Pol and Env), but it
30 species of non-human primates in sub-Saharan Africa is not known which (if any) of these differences are relevant
(Bibollet-Ruche et al. 2004a). These simian immunodeficiency
to the biology of these viruses in the two different hosts.
viruses (SIVs) form host-species specific clades, and in
Although the first SIVcpz isolate was described more
their natural hosts appear to be nonpathogenic. HIV-AIDS than 15 years ago (Peeters et al. 1989), by 2003 only four
emerged in the 20th century after humans acquired SIVs
full-length sequences of SIVcpzPtt had been determined
from two different species, chimpanzees (Pan troglodytes)
because captive chimpanzees are only rarely infected with
and sooty mangabeys (Cercocebus atys) (Sharp et al. 2001).
this virus (Sharp et al. 2005). We have since sequenced
The vast majority of HIV-AIDS cases around the
eight additional SIVcpzPtt strains, including viruses that
world are due to human immunodeficiency virus type 1
represent the closest known relatives of HIV-1 groups M
(HIV-1). HIV-1 originated from cross-species transmis-
and N (Bibollet-Ruche et al. 2004b; Nerrienet et al. 2005;
sions of SIVcpzPtt infecting chimpanzees (P. t. troglo-
Keele et al. 2006; Van Heuverswyn F, unpublished data).
dytes) in west central Africa (Gao et al. 1999). HIV-1
Thus, there are now twelve full length SIVcpzPtt sequences
strains are classified into three distinct groups (M, N
available, as well as four of SIVcpzPts from P. t. schwein-
and O) which are interspersed among SIVcpzPtt lineages furthii which form an outgroup to the SIVcpzPtt/HIV-1
in phylogenetic trees (Fig. 1), indicating that they arose
clade. This provides an opportunity to look for viral genetic
from three independent ape-to-human transmissions (Hahn
changes associated with cross-species transmission that may
et al. 2000). The three transmissions have had very differ-
represent adaptations of SIVcpz to its new human host. Here
ent outcomes: group N strains have been found in only
we identify one such site, in the p17 matrix protein encoded
a limited number of individuals in Cameroon, group O
by the gag gene, which shows clear evidence of having
strains are more widespread but mainly restricted to indi-
evolved under host-specific selection pressure. Further-
viduals from Cameroon and surrounding countries, while
more, we demonstrate that replacements at this site have
group M strains have spread throughout Africa and the rest
different effects on virus replication in CD4þ T-cells from
humans and chimpanzees.
Key words: HIV-1, SIV, matrix protein, cross-species transmission,
host-specific adaptation. Materials and Methods
E-mail: paul.sharp@ed.ac.uk. Sequence Analyses
Mol. Biol. Evol. 24(8):1853–1860. 2007
doi:10.1093/molbev/msm110 Protein sequences were aligned using ClustalW
Advance Access publication June 1, 2007 (Thompson et al. 1994), with minor manual adjustment.
Ó The Author 2007. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org
1854 Wain et al.
SIVcpzMB897
SIVcpzMB66
SIVcpzLB7
HIV-1 M
HIV-1 M
SIVcpzEK505
HIV-1 N
HIV-1 N
SIVcpzMT145
SIVcpzCAM5
SIVcpzCAM3
SIVcpzDP943
SIVcpzUS
SIVcpzCAM13
SIVcpzGAB1
SIVcpzGAB2
HIV-1 O
HIV-1 O
SIVgor
SIVcpzTAN1
SIVcpzTAN2
SIVcpzTAN3
SIVcpzANT
FIG. 1.—The three independent origins of HIV-1. Red arrows
indicate the branches on which ape-to-human transmissions can be
inferred to have occurred, and black circles denote the ancestors of HIV-1
groups M, N and O. Colours indicate SIVcpz strains from Pan
troglodytes troglodytes (red) and P. t. schweinfurthii (blue). The tree
represents a strict consensus of phylogenies derived from four major
regions of the proteome (Keele et al. 2006). The evolutionary relation-
ships among SIVcpzPtt strains, and among SIVcpzPts strains, vary
depending on the region of the genome used for analysis, presumably
reflecting recombination events in the past. However, the distinction
between the SIVcpzPtt and SIVcpzPts lineages, and the positions of HIV-
1 groups M (close to SIVcpzPtt strains LB7, MB66 and MB897), N
(close to SIVcpzPtt strain EK505) and O (as an outgroup to the known
SIVcpzPtt strains) are constant. The dashed line indicates the position of
SIVgor based on partial sequences (Van Heuverswyn et al. 2006); note
that HIV-1 group O may have been transmitted from chimpanzees to
humans via gorillas.
Phylogenetic relationships were inferred by the Bayesian
method (Yang and Rannala 1997) implemented in MrBayes
(Huelsenbeck and Ronquist 2001), using the JTT model of
amino acid substitution (Jones et al. 1992) and a gamma
distribution of among-site rate variation with four categories
(Yang 1994); analyses were run for 1 million generations.
The HIV-1 group M ancestral sequence was obtained
from the HIV Sequence Database (www.hiv.lanl.gov). The
group O ancestral sequence was inferred from 13 full length
genome sequences; sequences previously shown to be re-
combinant (Yamaguchi et al. 2003) were excluded from
this analysis. (Accession numbers of all sequences used
are provided in the Supplement.) A phylogeny estimated
from concatenated Gag, Pol and Env sequences, rooted
by reference to SIVcpzPtt sequences, was used to find
the maximum likelihood estimate of the ancestral group
O sequence using Codeml from the PAML package (Yang
1997), under the assumption of a molecular clock using the
JTT matrix and gamma distributed rates at sites. The HIV-1
group N ancestral sequence was obtained by a similar anal-
ysis of five genome sequences, with SIVcpzPtt/EK505 used
to root the phylogeny.
We searched for species-specific signatures in align-
ments of Gag, Pol, Vif, Vpr, Vpu, Tat, Rev and Nef pro-
tein sequences of 19 viral strains (twelve SIVcpzPtt, four
SIVcpzPts, and the three HIV-1 group ancestral sequences).
We were looking for sites that are highly conserved among
SIVcpzPtt strains, but since any of the SIVcpz sequences
may have been derived from a defective viral genome,
we allowed for some deviation from perfect conservation
among all sequences; this same concern does not apply
to HIV-1 group ancestor sequences. Thus, we searched
for sites where the amino acid was conserved among at least
nine of the twelve SIVcpzPtt sequences, and identical
among all three HIV-1 group ancestors, but differed be-
tween HIV-1 and the consensus sequence of SIVcpzPtt.
The degree of biochemical dissimilarity among amino
acids was assessed by reference to Grantham’s chemical
distances. These distances combine information on chem-
ical composition, polarity and molecular volume, and are
highly correlated with amino acid replacement frequencies
(Grantham 1974). The values for the 190 pair-wise compar-
isons among the 20 amino acids range from 5 to 215, with
a mean of 100.
Cell Lines, Viruses and Virus Stocks
293T and JC53BL13 cells were maintained in Dulbec-
co’s modified Eagle’s medium (DMEM) þ 10% heat inac-
tivated fetal bovine serum (FBS). Replication competent
molecular clones of chimpanzee-passaged HIV-1 (JC16
and NC7) were obtained from Dr. Novembre (Mwaengo
and Novembre 1998). Viral stocks of JC16 and NC7 were
generated by transfecting full-length proviral plasmids into
293T cells using FuGene 6 (Roche Applied Science,
Indianapolis, IN). Seventy-two hours post-transfection,
virus-containing supernatants were clarified by low speed
centrifugation and frozen in aliquots at -70C. The infectivity
of virus stocks was determined using the JC53BL13 assay as
described previously (Derdeyn et al. 2000; Wei et al. 2002).
To change the amino acid at Gag-30 in JC16 and NC7
from a Met to a Lys, site-directed mutagenesis was per-
formed using the QuickChange II XL kit (Stratagene, La
Jolla, CA) and the primers JCNC-M30K-F (5’-GGAAA
GAAAAAATATAAGTTAAAACATATAGTATGG-3’)
and JCNC-M30K-R (5’-CCATACTATATGTTTTAACT
TATATTTTTTCTTTCC-3’).
Plasmids were grown in STBL2 bacteria (Invitrogen,
Carlsbad, CA) as described by Takehisa et al. (2007).
Isolation and Activation of Chimpanzee and Human
PBMCs
Blood was obtained from healthy HIV-1-negative hu-
man volunteers (Research Blood Components, Boston,
MA), as well as HIV/SIV-uninfected chimpanzees housed
at the Yerkes Primate Research Center in accordance with

Animal Welfare Act guidelines. Chimpanzee blood sam-
ples were collected in tubes containing acid citrate dextrose
from anaesthetized individuals during their annual health
survey (this procedure was approved by the Emory Institu-
tional Animal Care and Use Committee). Both human and
chimpanzee peripheral blood mononuclear cells (PBMCs)
were isolated by density separation using Ficoll-hypaque
plus (GE-Healthcare, Piscatawy, NJ) and centrifuged at
1800 rpm for 25 min at 22°C. Buffy coat cells were washed
once at room temperature in Hanks Balanced Saline Solu-
tion (HBSS) þ 4 mM EDTA and once at 4°C in HBSS þ
1% FBS. CD4þ T lymphocytes were enriched using CD4-
containing microbeads by magnetic cell sorting according
to the manufacturer’s protocols (Militenyi Biotec, Auburn,
CA). For activation, CD4þ T lymphocytes were allowed to
adhere for 30 minutes and then stimulated for 12-15 hours
with 3lg/ml of Staphylococcal Enterotoxin B (Sigma-
Aldridge, St. Louis, MO) in RPMI þ 15% FBS. Suspension
cells were washed once with HBSS, resuspended in DMEM
with 10% GCT conditioned media (BioVeris Corp.,
Gaithersburg, MD) and 10% Human AB serum (Fisher
Bioreagents, Fair Lawn, NJ), returned to the same well
in which they were activated, and incubated for 5-6 days
at 37°C in a humidified 5% CO2 incubator. Suspension cells
were then resuspended at 1 x 106 cells /ml in DMEM with
10% FBS and 30 IU of IL-2/ml (Roche Applied Science,
Indianapolis, IN) for 24 hours prior to infection.
Replication Kinetics
Approximately 500,000 activated human or chimpan-
zee CD4þ T lymphocytes were infected overnight at a mul-
tiplicity of infection of 0.01 (JC53BL titer) in 300ll of
DMEM þ 10% FBS þ 30 units IL-2/ml. The following
morning cells were washed three times with HBSS and
plated in 24 well plates in 2 ml of DMEM þ 10%
FBS þ 30 units of IL-2/ml. Forty microliters of supernatant
were collected every other day and stored at -70°C. Cultures
were maintained for 9 days. Virus replication was assessed
by quantifying the amount of p24 core protein released into
the culture supernatant using the HIV-1 p24 antigen EIA kit
(Beckman Coulter, Fullerton, CA) according to the manu-
facturer’s protocol.
Experiments involving pairs of viruses, with either
Met or Lys at Gag-30, were performed in cells from the
same donors. Each experiment was repeated four times.
The significance of the higher growth rate of Met-encoding
viruses in chimpanzee cells, or of Lys-encoding viruses in
human cells, was assessed in a one-tailed paired t-test of
log-transformed data from day 9.
Results
Identification of a Host-Specific Substitution in HIV-1
We compared the inferred ancestral sequences at the
root of each HIV-1 group to SIVcpz sequences, seeking
changes that had occurred in parallel on each branch of
the tree where cross-species transmission from chimpan-
zees to humans occurred (fig. 1). SIVcpz/HIV-1 genomes
Adaptation of HIV-1 to its human host 1855
Table 1
Sites Differing Between HIV-1 and SIVcpzPtt
Sitea HIV-1 SIVcpzPtt SIVcpzPts HIV-1/cpz
Gag-30 Argb Met(12)c Leu(3), Met(1) Met
Gag-224 Pro Ala(9), Pro(3) Gln(3), Pro(1) Ala
Gag-335 Lys Arg(10), Lys(2) Lys(4) Lys
Pol-175 Lys Arg(9), Lys(3) Lys(3), Arg(1) Lys
Pol-497 Tyr Phe(9), Tyr(3) Tyr(4) Tyr
Vif-185 Gly Glu(9), Gly(3) Glu(2), Gly(2) Gly
Nef-46 Ser Arg(11), Ser(1) Asn(3), Ser(1) Ser
NOTE.— HIV-1 refers to the inferred ancestral sequences of the three groups of
HIV-1. HIV-1/cpz refers to the sequence of HIV-1 strains after passage in
chimpanzees for more than 10 years.
a
sites numbered according to the reference strain HIV-1/HXB2.
b
amino acid found in HIV-1 in bold.
c
numbers of occurrences among SIVcpz sequences in brackets.
encode nine proteins. We searched alignments of all nine
proteins for sites that are conserved among SIVcpzPtt
strains but differ (with the same change) in all three ances-
tral HIV-1 sequences. An analysis allowing up to three of
the 12 SIVcpzPtt sequences to differ from the consensus
sequence identified seven sites from four different proteins
(table 1). Only one of these sites (Gag-30) was completely
conserved among the 12 SIVcpzPtt sequences. At all six
other sites the amino acid found in HIV-1 was present as
a minor variant in SIVcpzPtt and was also present in
SIVcpzPts; at all six sites the amino acid found in HIV-1
was present in at least two SIVcpz sequences (SIVcpzPtt
or SIVcpzPts). Thus, there was only one site which was
highly conserved among SIVcpz sequences and yet con-
tained the same replacement in all three HIV-1 group an-
cestors, with an amino acid not found in any of the SIVcpz
sequences. Gag-30 is Met in all 12 SIVcpzPtt sequences
and in one SIVcpzPts, Leu in three (closely related)
SIVcpzPts, and Arg in all three HIV-1 group ancestors
(fig. 2). This implies that the ancestor of the entire SIVcpz
clade had Met at this position, which has subsequently been
highly conserved among chimpanzee viruses, undergoing
a biochemically conservative change to Leu (Grantham’s
amino acid distance, D 5 15) only in one lineage within
SIVcpzPts. However, on all three branches leading to
the HIV-1 groups (fig. 1), Met has been replaced by the
biochemically dissimilar, basic amino acid Arg (D 5 91).
The available strains of SIVcpzPtt include three very
closely related to HIV-1 group M, as well as one very
closely related to group N (fig. 3; see also Keele et al.
2006). These strains serve to locate two of the Met-to-
Arg changes on relatively short branches immediately prior
to the ancestral nodes in groups M and N. As yet no
SIVcpzPtt strain has been found that is very closely related
to HIV-1 group O (fig. 3). However, we recently reported
the discovery of SIV in gorillas (Van Heuverswyn et al.
2006); on the basis of partial pol and env sequences, these
SIVgor viruses are closely related to HIV-1 group O (fig. 1).
We have subsequently obtained a partial gag sequence for
one SIVgor strain (F. Van Heuverswyn, unpublished). Like
the SIVcpzPtt sequences, this SIVgor sequence encodes
Met at Gag-30, placing the third Met-to-Arg change on
the branch after the split of the ancestors of SIVgor and
HIV-1 group O.
1856 Wain et al.

SIVcpzMB897
100
SIVcpzMB66
80
88 SIVcpzLB7
100 100 HIV-1 M
HIV-1 M
SIVcpzEK505
100
100 HIV-1 N
99
HIV-1 N
SIVcpzMT145
100
SIVcpzCAM5
86 SIVcpzCAM3
99
100 SIVcpzDP943
100
100 SIVcpzUS
100 SIVcpzCAM13
SIVcpzGAB1
96 SIVcpzGAB2
100 HIV-1 O
HIV-1 O
SIVcpzTAN1
100 SIVcpzTAN2
FIG. 2.—Species-specific adaptive changes in the matrix protein SIVcpzTAN3
(Gag p17). Sequences from a region in the N-terminal basic domain of SIVcpzANT
0.05
HIV-1/SIVcpz matrix proteins reveal a site (boxed) that differs between
the HIV-1 group ancestors (R, Arg) and chimpanzee viruses (M, Met/L, FIG. 3.—Phylogeny of the SIVcpz/HIV-1 clade derived from anal-
Leu). Dashes indicate amino acid identity to the sequence at the top. ysis of Gag protein sequences. SIVcpzPtt and SIVcpzPts strains are in red
Clones JC16 and NC7 were isolated from two chimpanzees experimen- and blue, respectively. The representative HIV-1 sequences used were
tally infected with HIV-1 (Mwaengo and Novembre 1998). U455 and LAI (group M), YBF30 and YBF106 (group N) and ANT70
and MVP1580 (group O). A posteriori probabilities (expressed as percent)
are shown above branches. The scale bar represents 0.05 amino acid
To assess how unusual the observation at Gag-30 is, replacements per site.
we searched for any sites in the Gag alignment where,
within the SIVcpzPtt/HIV-1 clade, any three sequences
shared one residue while all 12 other sequences shared an- ical distances than the change at Gag-30 (table 2). In light of
other residue; in addition, we required that the residue these data, the finding of a non-conservative Met-to-Arg
found in the three sequences should not be present in change on all three (and only those three) branches leading
any of the SIVcpzPts sequences. After excluding sites with to the human viruses is quite remarkable and highly un-
a gap in any sequence, the alignment contained 450 sites. likely to represent a chance occurrence.
This analysis identified six sites, including Gag-30, the site
already found (table 2). However, consideration of the phy-
logenetic relationships among the gag genes of these viral Evolution of HIV-1 in Experimentally
strains (fig. 3) indicates that four of these sites require fewer Infected Chimpanzees
than three independent substitutions. SIVcpzPtt strains To investigate further whether the observed replace-
GAB1 and CAM13 form a monophyletic pair, and replace- ment at Gag-30 was indicative of host-specific adaptation,
ments at Gag-19 and Gag-380 likely occurred in their com-
mon ancestor. Similarly, a replacement at Gag-52 likely
occurred in the common ancestor of SIVcpzPtt strain Table 2
EK505 and HIV-1 group N. Thus, these three sites require Sites in Gag Differing between Three HIV-1/SIVcpzPtt
only two independent substitutions. Only one replacement Sequences and All Others
is required at site Gag-231, since SIVcpzPtt strains CAM3, Sitea Set 1b AA Set 2b D1c SIVcpzPts D2c
CAM5 and DP943 form a monophyletic clade (fig. 3). The
remaining site, Gag-28, would require three independent Gag-19 GAB1, CAM13, LB7 Val Ile 29 Ile -
Gag-28 GAB1, GAB2, LB7 Arg Lys 26 Lys -
replacements, although it is equally parsimonious to invoke Gag-30 M, N, O Arg Met 91 Met/Leu 15
three independent Lys-to-Arg changes on terminal branches Gag-52 EK505, MB897, N Asp Glu 45 Glu -
of the tree, or two Lys-to-Arg changes and a reversion of Gag-231 CAM3, CAM5, DP943 Ala Pro 27 Pro -
Arg-to-Lys on the terminal branch to CAM13. Thus only Gag-380 GAB1, CAM13, US Lys Arg 26 Arg/Gly 125
one other site, in addition to Gag-30, was found that a
sites numbered according to the reference strain HIV-1/HXB2.
may have undergone the same replacement on three termi- b
Set 1 are the three sequences from the SIVcpzPtt/HIV-1 clade sharing the
nal branches of the tree. These replacements involving ba- unusual residue; M, N and O refer to HIV-1 groups, while the other sequences are
sic residues at Gag-28 are much more conservative (D 5 all SIVcpzPtt strains. Set 2 shows the amino acid found in the remaining 12
sequences from the SIVcpzPtt/HIV-1 clade.
26) than the Met-to-Arg change at Gag-30, and likely reflect c
D values are Grantham’s amino acid distances. D1 is the distance between
recurrent neutral switches. The other sites identified in this the residues found in Set1 and Set 2. D2 is the distance between the two residues
analysis also involve amino acids with much smaller chem- found in SIVcpzPts.
 Adaptation of HIV-1 to its human host 1857

FIG. 4.—Replication of variants of JC16 (upper) and NC7 (lower) strains in chimpanzee (left) and human (right) CD4þ T lymphocytes. For each
virus, strains encoding Met (blue) or Lys (red) at Gag-30 are compared. Virus replication was monitored by measuring the level of the p24 antigen in
culture supernatants (note that the scale varies between left and right panels). Replication curves are shown as the average of four independent
experiments in CD4þ T lymphocytes from different chimpanzee and human donors; the error bars represent one standard deviation calculated for each
time point.

we examined the sequences of two molecular clones (JC16
and NC7) that were recovered from chimpanzees experi-
mentally inoculated with HIV-1 (Mwaengo and Novembre
1998). The rationale for these chimpanzee infections was to
generate pathogenic variants for vaccine and pathogenesis
studies (Novembre et al. 1997; Wei and Fultz 1998). Al-
though chimpanzees were subsequently found not to repre-
sent a viable animal model, the viral isolates and molecular
clones derived from these experiments are of value because
they represent chimpanzee-adapted strains of HIV-1. Clone
JC16 was recovered from a chimpanzee 10 years after his
initial infection; clone NC7 was recovered from a second
chimpanzee one month after receiving blood from the first
chimpanzee (Mwaengo and Novembre 1998). The two
clones differ at 1.4% of nucleotides; given the rate of
HIV-1 evolution (Li et al. 1988), this indicates that they
may have diverged about 1-2 years before they were iso-
lated. These nucleotide substitutions cause 51 amino acid
differences across the viral proteome. To determine whether
prolonged propagation in chimpanzees had exerted selec-
tion pressure on the Gag-30 residue, we compared the se-
quences of JC16 and NC7 to their parental strains, which
represented a mixture of group M subtype B strains (SF2
and LAV) and thus contained a conservative replacement
of Arg with Lys at Gag-30 (Wei and Fultz 1998). Remark-
ably, inspection of JC16 and NC7 revealed Met at Gag-30
in both clones, indicating selection pressure for this residue
in the chimpanzee host (fig. 2).
The six other sites initially identified as differing
between HIV-1 and the majority of SIVcpzPtt strains all
retained the HIV-1 residue after passage in chimpanzees
(table 1). At one site, Gag-224, the residue in the
chimpanzee-adapted virus (Ala) differed from that in the
three HIV-1 group ancestor sequences (Pro). However, this
site is Ala in most HIV-1 subtype B sequences, including
the viruses used in the chimpanzee passage experiment.
Replication of JC16 and NC7 in Chimpanzee and
Human T Cells
To assess whether replacements at Gag-30 confer
a host species specific growth advantage, we applied site-
directed mutagenesis to the chimpanzee adapted JC16
and NC7 clones to change the Met back to Lys, and as-
sessed their replication potential in CD4þ T lymphocytes
from multiple chimpanzee and human donors. These ex-
periments revealed a clear and reproducible correlation be-
tween the identity of the amino acid at Gag-30 and the
ability of viruses to replicate in cells from the two different
species (fig. 4). In chimpanzee cells, JC16 and NC7 with
Met at Gag-30 (blue) grew, on average, to much higher
1858 Wain et al.
titers than the same viruses with Gag-30 mutated to Lys
(red). However, there was considerable variation among
replicates: in tests of the day 9 titers, NC7 with Met was
significantly higher than NC7 with Lys (P , 0.05), but
for JC16 the comparison only bordered on significance
(P 5 0.054). In human cells, these chimpanzee adapted vi-
ruses exhibited low replicative fitness (compare the scales
in the left and right panels of fig. 4), but both JC16 and NC7
viruses with Lys at Gag-30 replicated considerably more
efficiently than those with Met (P , 0.005, in both cases).
Evolution of Gag-30 in HIV-1
Arg at Gag-30 has remained highly conserved during
the diversification of HIV-1 groups M, N and O, typically
only being conservatively replaced by another basic amino
acid, Lys (D 5 26). Approximately 80% of available group
O sequences have Arg, while in the other 20% it is Lys.
Only six group N virus Gag sequences have thus far been
characterized: five have Arg, and one has Lys. Among the
group M subtype consensus sequences compiled at the HIV
Sequence Database (www.hiv.lanl.gov), Arg is present in
subtypes A1, A2, D, F1, G, H and K, while the subtype
B consensus sequence has Lys. Interestingly, the only
HIV-1 group M subtype that does not contain a basic res-
idue at Gag-30 is subtype C. Met is found in both the con-
sensus sequence and the inferred ancestral sequence of
subtype C. Since subtype C is not the basal lineage in
the group M radiation (Korber et al. 2000; Leitner et al.
2005), this indicates that reversion of Arg to Met occurred
subsequent to the divergence of the subtype C ancestor
from the other group M lineages.
Discussion
Comparisons of the inferred sequences of the ances-
tors of the three independent groups of HIV-1 with those
of SIVcpz strains revealed a single site in the HIV-1 pro-
teome which appears to have undergone identical changes
on each of the three occasions when virus was transmitted
from apes to humans. All available sequences of SIVcpzPtt,
the clade from within which HIV-1 originated, have Met at
Gag-30, while the ancestors of all three HIV-1 groups had
Arg. Gag-30 was the only site, among 450 in the Gag align-
ment, to show the same non-conservative amino acid re-
placement on three independent terminal branches within
the SIVcpzPtt/HIV-1 clade, and so it is remarkable that
these changes were found specifically on the branches lead-
ing to the three HIV-1 groups. The finding of this non-
conservative amino acid replacement on the branches of
the tree immediately prior to each of the three clades of hu-
man viruses, at a site which is highly conserved among
chimpanzee viruses, strongly suggests host-species specific
adaptation. During the subsequent spread and evolution of
HIV-1 in humans, Gag-30 has been conserved as Arg, or
conservatively replaced by another basic amino acid
(Lys), in all lineages except group M subtype C.
Retrospective analysis of the result of passaging HIV-
1 in chimpanzees for more than 10 years (Mwaengo and
Novembre 1998) revealed that the basic residue at Gag-
30 had reverted to Met, providing independent evidence
of host-species specific selection on this site. Thus, while
the Met-to-Arg change on the lineages leading to HIV-1
could have occurred prior to cross-species transmission,
possibly predisposing particular chimpanzee viruses to
greater fitness in humans, the reversion to Met in chimpan-
zees infected with HIV-1 argues that the adaptive changes
occurred after the cross-species transmission events.
Previously it has been shown that mutation of HIV-1
subtype B Gag-30 to a non-basic residue (Thr), in combi-
nation with a similar mutation at Gag-32, greatly reduced
viral replication (Freed et al. 1995). Here we have found
that when the Met at Gag-30 in two chimpanzee-adapted
HIV-1 strains was replaced by Lys, the growth rate of these
viruses in chimpanzee CD4þ T cells was reduced by half.
In contrast, in human cells these same strains with Lys grew
twice as efficiently as strains with Met. These reciprocal
differences in replication capacity confirm that the nature
of the amino acid at Gag-30 can have a major effect on viral
fitness in cells from different host species. However, the
greatly reduced growth rate of the chimpanzee-adapted vi-
ruses in human T cells, even after the replacement of Met
with Lys at Gag-30, indicates that additional sites have
evolved away from being human-adapted.
In this context, it is intriguing that in subtype C, a major
sublineage within HIV-1 group M, Gag-30 has reverted to
Met. Subtype C is very widespread: it is the predominant
form of HIV-1 in southern Africa and also responsible for
AIDS epidemics in China and India (Hemelaar et al. 2006).
It is thus clear that HIV-1 strains with Met at Gag-30 can be
epidemiologically successful. However, subtype C isolates
have been shown to replicate less efficiently than other sub-
types in head-to-head ex vivo competition experiments in
primary CD4þ T cells and macrophages (Ball et al.
2003; Arien et al. 2005). This finding has been interpreted
to indicate that disease progression may be slower in sub-
type C infections, providing a longer period when infected
individuals may transmit virus to others and thus explaining
why subtype C is so common globally (Arien et al. 2007).
Whether there is indeed a causal link between the observed
in vitro phenotype of subtype C and its ability to spread in
the human population requires further studies, especially in
light of data indicating that in vivo viral loads are higher
in subtype C infections (Dyer et al. 1998). However, it is
tempting to speculate that the reduced replication fitness
of subtype C viruses in primary lymphocyte cultures may
be due, at least in part, to the Met residue at Gag-30. Since
the host-specificity at this site is otherwise so consistent, it
is also possible that the ancestral subtype C lineage under-
went changes at other sites which removed the need for, and
perhaps even selected against, a basic residue at Gag-30
during replication in human cells. A number of subtype
C viruses have Arg at Gag-30, but we could not find any
meaningful correlation between the presence or absence
of Met at Gag-30 and any polymorphisms at other sites. Site
directed mutagenesis of prototypic subtype C strains, as re-
ported here for chimpanzee adapted subtype B strains, may
shed further light on these possibilities.
The residue present at Gag-30 varies among SIVs in-
fecting different primate species. Interestingly, it is also Met
in SIVsmm from sooty mangabeys, the progenitor of HIV-2.

Eight different groups of HIV-2 have been described, which
each seem to reflect an independent cross-species transmis-
sion event (Hahn et al. 2000; Damond et al. 2004; Santiago
et al. 2005), although only two of these groups (A and B)
are known to have spread in the human population. Re-
markably, this residue changed to Arg in the inferred ances-
tor of the most widespread form of HIV-2, group A.
However, it has been conserved as Met in HIV-2 group
B, and was not found to have changed in any of the sporadic
forms of HIV-2. These observations provide additional ev-
idence that a basic residue at Gag-30 enhances the replica-
tion potential and possibly the secondary spread of primary
lentiviruses after transmission to humans.
Gag-30 lies within the basic N-terminal domain of the
gag-encoded matrix protein (p17). This domain is critical
for targeting the Gag precursor to the plasma membrane
during virus assembly (Hill et al. 1996). Several cellular
factors are known (or believed) to bind this same region.
These include PI(4,5)P2, a plasma membrane component
(Ono et al. 2004), TIP47, a recently discovered Gag/Env
connector protein complex (Lopez-Verges et al. 2006),
and AP-3, an adaptor protein complex that is involved in
cargo protein sorting to specific membrane components
(Dong et al. 2005). NMR studies have shown that Gag-
30 is not a part of the hydrophobic cleft that binds
PI(4,5)P2 (Saad et al. 2006). The TIP47 binding site on
the HIV-1 matrix protein has not yet been mapped; how-
ever, mutagenesis studies suggest that two highly con-
served matrix regions (S6-S9 and D14-E17) are likely
involved (Lopez-Verges et al. 2006). Finally, the d subunit
of the AP-3 complex is believed to interact with the amino-
terminal a-helical segment of the matrix protein (Dong et al.
2005). Thus, there are a number of candidate human pro-
teins that could bind the SIVcpz matrix proteins with re-
duced affinity due to species specific differences in their
protein sequence, and the same could be true for chimpanzee
proteins and their interaction with the HIV-1 matrix protein.
In conclusion, although the specific function of Gag-
30 remains to be determined, it is clear that adaptive
changes at this residue in the SIVcpz matrix protein were
required for efficient replication in the human host. These
findings represent the first demonstration of host species-
specific selection pressure in primate lentiviruses and iden-
tify the Gag matrix protein as a modulator of virus fitness
following cross-species transmission to a new primate host.
Supplementary Material
A list of the GenBank accession numbers of all se-
quences analyzed is available at Molecular Biology and
Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
We thank Wes Sundquist, Eric Freed, Mike Emerman,
Heinrich Gottlinger and Mike Summers for unpublished in-
formation, and Mike Worobey and John Brookfield for dis-
cussion. This work was supported by the National Institutes
of Health (R01 AI50529, R01 AI58715, P30 AI 27767, P30
Adaptation of HIV-1 to its human host 1859
CA 13148), the Yerkes Regional Primate Research Center
(RR-00165), the Bristol Myers Freedom to Discover Pro-
gram, and the Institut de Recherche pour le Développement
(IRD).
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Robin Bush, Associate Editor
Accepted May 22, 2007