Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env.
ABSTRACT Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4(+) T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a naïve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-kappaB binding site was engineered into its 3' long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.
- SourceAvailable from: Derya Unutmaz[show abstract] [hide abstract]
ABSTRACT: Entry of HIV-1 into target cells requires cell-surface CD4 and additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T-cell lines was recently identified and named fusin. However, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains in vivo. The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the beta-chemokines RANTES, MIP-1alpha and MIP-1beta.Nature 07/1996; 381(6584):661-6. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruses.Nature 07/1996; 381(6584):667-73. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Genetic variants of human and simian immunodeficiency virus (HIV and SIV) that evolve during the course of infection and progression to AIDS are phenotypically and antigenically distinct from their progenitor viruses present at early stages of infection. However, it has been unclear how these late variants, which are typically T-cell tropic, cytopathic and resistant to neutralizing antibodies, influence the development of clinical AIDS. To address this, we infected macaques with cloned SIVs representing prototype variants from early-, intermediate- and late-stage infection having biological characteristics typical of viruses found at similar stages of HIV infection in humans. These studies demonstrate that sequential, phenotypic and antigenic variants represent viruses that have become increasingly fit for replication in the host, and our data support the hypothesis that emerging variants have increased pathogenicity and drive disease progression in SIV and HIV infection.Nature Medicine 06/1999; 5(5):535-41. · 22.86 Impact Factor
JOURNAL OF VIROLOGY, Sept. 2006, p. 8729–8738
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 17
Molecularly Cloned SHIV-1157ipd3N4: a Highly Replication-
Competent, Mucosally Transmissible R5 Simian-Human
Immunodeficiency Virus Encoding HIV Clade C env
R. J. Song,1,2A.-L. Chenine,1,2R. A. Rasmussen,1,2C. R. Ruprecht,1S. Mirshahidi,1,2R. D. Grisson,1,2
W. Xu,1,2J. B. Whitney,1,2L. M. Goins,1,2H. Ong,1P.-L. Li,1,2E. Shai-Kobiler,1,2T. Wang,1,2
C. M. McCann,1,2H. Zhang,3C. Wood,3C. Kankasa,4W. E. Secor,5H. M. McClure,6
E. Strobert,6J. G. Else,6and R. M. Ruprecht1,2*
Dana-Farber Cancer Institute, Boston, Massachusetts 021151; Harvard Medical School, Boston, Massachusetts 021152;
Nebraska Center for Virology and School of Biological Science, University of Nebraska, Lincoln, Nebraska 685883;
University Teaching Hospital, Lusaka, Zambia4; Division of Parasitic Diseases, Centers for Disease Control
and Prevention, Atlanta, Georgia 303415; and Yerkes National Primate Research Center,
Emory University, Atlanta, Georgia 303226
Received 16 March 2006/Accepted 16 June 2006
Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and
an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human
immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS
vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian
infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three
of which developed peripheral CD4?T-cell depletion. After the first inoculated monkey developed AIDS at
week 137 postinoculation, transfer of its infected blood to a naı ¨ve animal induced memory T-cell depletion and
thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was
amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-
1157ipd3, an extra NF-?B binding site was engineered into its 3? long terminal repeat, giving rise to SHIV-
1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood
mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of
Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated
vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent,
mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting
HIV-1 clade C Env.
Simian-human immunodeficiency virus (SHIV) strains are
chimeric viruses constructed from the pathogenic simian im-
munodeficiency virus (SIV) clone SIVmac239, in which the
env, tat, and rev genes had been replaced by the corresponding
regions of human immunodeficiency virus type 1 (HIV-1);
most SHIVs also contain vpu (7, 21, 22, 25, 27, 31, 35, 47).
SHIV infection of rhesus macaques has been an invaluable in
vivo model to study the role of HIV-1 envelopes in transmis-
sion and pathogenesis as well as to evaluate the efficacy of
candidate vaccines based on HIV-1 envelope glycoproteins,
which specify cell tropism and coreceptor usage and are also
primary targets of the immune response. However, the major-
ity of current SHIV strains utilize envelope genes derived from
HIV-1 clade B strains, which represent less than 10% of all
global infections. Therefore, the available SHIV chimeras do
not reflect the genetic diversity of the HIV-1 epidemic, which
is dominated by non-B clades, especially HIV-1 clade C.
HIV-1 clade C, the dominant subtype in the world, is esti-
mated to comprise more than 50% of all infections in the
pandemic and is the most prevalent clade in sub-Saharan Af-
rica and parts of Asia, where the AIDS epidemic is growing
fastest (http://www.unaids.org). The rapid spread of this par-
ticular subtype in these heavily populated regions has resulted
in over 5 million infections in Asia alone.
Over 90% of all HIV transmission events worldwide involve
mucosal transmission, including most sexual and mother-to-
child transmissions (45). HIV-1 strains isolated from individ-
uals soon after infection (14, 34, 59) preferentially use CCR5
as the coreceptor for cell entry (2, 12, 15, 18). Such viruses are
referred to as R5 HIV-1 isolates. Therefore, a highly replica-
tion-competent SHIV that is mucosally transmissible in rhesus
monkeys and that encodes a non-clade B HIV-1 env gene
would be an important tool in HIV/AIDS research.
Although several non-clade B HIV-1 envelope-based SHIV
chimeric constructs have been described so far (8, 10, 30, 41,
58), none of them has been reported to be mucosally trans-
missible or to induce signs of disease in rhesus macaques, the
most commonly used nonhuman primate in AIDS research.
Here we report the construction of SHIV-1157ipd3N4. This
virus was isolated from a rhesus monkey, RPn-8, which had
been inoculated as an infant with a parental SHIV construct
that expresses the envelope glycoprotein of a relatively recently
transmitted R5 HIV-1 clade C isolate from a 6-month-old
Zambian infant. SHIV-1157ipd3N4 was derived from this
* Corresponding author. Mailing address: 44 Binney St., Dana-Far-
ber Cancer Institute, JFB809, Boston, MA 02115. Phone: (617) 632-
3719. Fax: (617) 632-3112. E-mail: firstname.lastname@example.org.
same animal, RPn-8, after it developed AIDS approximately
2.7 years postinoculation. SHIV-1157ipd3N4 exclusively uses
CCR5 as a coreceptor and could be intrarectally transmitted to
rhesus monkeys of both Indian and Chinese origin.
MATERIALS AND METHODS
Original virus isolates and nomenclature. HIV1157i is a biological isolate
obtained from a Zambian infant at 6 months of age. At birth, this infant was PCR
positive for HIV-1. The designation “i” indicates a virus strain (or env gene)
isolated from an infant. SHIV-1157i is the original infectious molecular clone,
not yet adapted to rhesus monkeys. SHIV-1157ip is an early biological isolate
obtained after passage through five rhesus monkeys; “p” designates a passaged
(or monkey-adapted) virus. SHIV-1157ipd is a late biological isolate; “d” indi-
cates that the virus was reisolated from an infected animal with disease (AIDS as
defined by persistent depletion of CD4?T cells to ?200 cells/?l). SHIV-
1157ipd3 designates the late-stage infectious molecular clone #3; the 3? half of
this provirus was derived from the biological isolate SHIV-1157ipd. SHIV-
1157ipd3N4 is identical to SHIV-1157ipd3 except that the 3? long terminal repeat
(LTR) was engineered to contain two rather than the usual one NF-?B site. This
NF-?B site duplication is copied into the 5? LTR during the reverse transcription
steps occurring in the course of the subsequent retroviral propagation (13).
Cell lines and antibodies. CEMx174-GFP cells, provided by B. Felber (Na-
tional Cancer Institute, Frederick, MD), contain the green fluorescent protein
gene under HIV-1 LTR regulation and express CXCR4 but not CCR5. U87 or
GHOST cell lines, which express CD4 only or CD4 with different chemokine
receptors, were provided by the AIDS Research and Reference Reagents Pro-
gram (ARRRP; Germantown, MD). TZM-bl cells (also called JC53-bl [clone 13]
cells; ARRRP) (16) are derived from a HeLa cell line (JC.53) that stably
expresses CD4 and CCR5. TZM-bl cells also express luciferase and ?-galacto-
sidase under control of the HIV-1 LTR. The neutralizing monoclonal antibody
(NMAb) 2G12 (57) was a gift of Hermann Katinger (Polymune Scientific, Vi-
Animals and animal care. Rhesus monkeys (Macaca mulatta) of Indian and
Chinese origin were used in this study. The animals were kept according to
National Institutes of Health guidelines on the care and use of laboratory ani-
mals at the Yerkes National Primate Research Center (Emory University, At-
lanta, GA) and the Centers for Disease Control and Prevention (CDC; Atlanta,
GA). These facilities are fully accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care International. Animal experiments
were approved by the Animal Care and Use Committees of the Yerkes National
Primate Research Center, the Centers for Disease Control and Protection, and
the Dana-Farber Cancer Institute.
Construction of SHIV-1157ipd molecular clones and sequence analysis. Using
a DNAzol genomic DNA isolation kit (Molecular Research Center Inc., Cincin-
nati, OH), chromosomal DNA was extracted from 106peripheral blood mono-
nuclear cells (PBMC) from animal RPn-8, the monkey first exposed to SHIV-
1157i, after its absolute CD4?T-cell counts fell below 200 cells/?l. A pair of
specific primers was designed to amplify the entire 3? half of SHIV-1157ipd. The
primers were designed to incorporate SphI or NotI restriction enzyme sites with
the following sequences: 1157ipd-SphI, 5?-CCGCCCTCTAGAAGCATGCTGT
AG-3?; and 1157ipd-NotI, 5?-AAAGTTGAATGCGGCCGCTACTTCTAAAA
TGGCAGCTTTATTGAAGAGG-3?. The 5? half of SHIV-vpu? (31) was di-
gested with the restriction enzymes EcoRI and SphI and cloned into vector
pSP73-N, which was modified with the introduction of an extra NotI site at the
multiple cloning sites of pSP73. The PCR products were digested with SphI and
NotI and cloned into the pSP73-N vector that has the 5? half of SHIV-vpu? to
form the full-length proviral DNA SHIV-1157ipd. The env gene of SHIV-
1157ipd was also amplified using primers 1157ipd-forward (5?-TACAAAGAG
GAAATGGATAAA-3?) and 1157ipd-reverse (5?-ATCCATGTGTGTACTATT
GTC-3?) and cloned into TOPO sequencing vector (Invitrogen, Carlsbad, CA).
Five clones were randomly picked for plasmid preparation and DNA sequencing.
An additional NF-?B element was added to the 3? LTR of SHIV-1157ipd3 using
the Quikchange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and a
pair of primers (N2-1, 5?-ACTCGCTGAAACAGCAGGGACTTTCCACAAA
GGGACTTTCCACAAGGGGATGTTACGGGGAGG-3?; and N2-2, 5?-CCTC
Construction of SIV LTR pLuc mutants. The pLuc reporter construct (a gift
of J. Clements, Johns Hopkins University, Baltimore, MD) (50) contains a
truncated portion of the SIVmac239 LTR (?2253?149), upstream of the firefly
luciferase reporter gene. HIV-1 or SIV Tat is required for the activation of pLuc
in order to drive expression of the luciferase gene by the LTR. The Quikchange
Site-Directed Mutagenesis kit (Stratagene) was used to introduce mutations into
the SIVmac239 LTR in the pLuc construct.
Coreceptor usage of SHIV constructs. The U87 or GHOST cell lines expressing
CD4 and/or HIV-1 or SIV coreceptors were used to study virus tropism. U87CD4,
U87CCR1, U87CCR2, U87CCR3, U87CXCR4, U87CCR5, GHOST.BOB, and
GHOST.BONZO were infected with 0.5 ml of virus stock. SHIVSF162P3was ob-
tained from ARRRP. Cells were washed and resuspended in 1 ml of fresh medium.
On days 1, 2, 3, 4, and 5, supernatants were collected for p27 measurement.
Measurement of plasma viral RNA levels. Plasma viral RNA was isolated by
use of the QiaAmp Viral Mini-kit (QIAGEN), and viral RNA levels were
measured by quantitative reverse transcriptase PCR (RT-PCR) for SIV gag
sequences (24) at weeks 0, 1, 2, 4, and 8 and monthly thereafter. The assay
sensitivities were 50 viral RNA copies/ml of plasma.
Generation of large-scale SHIV-1157ipd3N4 stock. A large-scale stock of the
infectious molecular clone was prepared by infecting concanavalin A (ConA)-
stimulated naı ¨ve rhesus monkey PBMC in the presence of human interleukin-2
(IL-2; 20 U/ml) and tumor necrosis factor alpha (TNF-?; 10 ng/ml) with virus
harvested from transiently transfected 293T cells. This rhesus PBMC-grown
stock has a p27 concentration of 227 ng/ml and 4 ? 10650% tissue culture
infectious doses (TCID50) per ml as titrated in TZM-bl cells.
TCID50determination of virus stock in TZM-bl cells. Viruses were added to
TZM-bl cells (16) at serial 1:4 dilutions in the presence of 40 ?g/ml of DEAE-
dextran hydrochloride (Sigma, St. Louis, MO). Virus infectivity was determined
48 h postinoculation by measuring the level of luciferase activity expressed in
infected cells. Each experiment was performed in triplicate. The TCID50was
calculated as the dilution point at which 50% of the cultures were infected.
Intrarectal inoculation of SHIV-1157ipd3N4. Chinese-origin rhesus monkeys
received 1 ml of the original large-scale virus stock intrarectally at various
dilutions: undiluted stock (one monkey), 1:5.5 (one monkey), 1:10 (one monkey),
and 1:50 (three monkeys; only two became systemically infected). Higher dilu-
tions of the stock did not result in systemic infection. Eight Indian-origin rhesus
macaques received 0.5 ml of the same large-scale virus stock intrarectally. These
monkeys were part of a DNA prime/protein boost vaccine study and served as
vector-only controls (R. A. Rasmussen, H. Ong, R. Song, E. Shai-Kolber, A.-L.
Chenine, S.-L. Hu, P. Policano, J. McKenna, J. Moon, B. Travis, H. M. McClure,
E. Strobert, F. J. Novembre, J. G. Else, and R. M. Ruprecht, unpublished data);
they had received 200 ?g of the empty DNA vector pJW4303 twice intradermally
at a 6-week interval as well as 0.1 ml of incomplete Freund’s adjuvant (IFA) twice
intramuscularly at a 6-week interval 1 year later. Two weeks after the second
inoculation of IFA, the animals underwent the intrarectal virus challenge. All
animals were monitored prospectively for viral loads and T-cell subsets at dif-
ferent time points postinoculation.
Neutralization assay. The neutralization assay was performed in triplicate in
human PBMC, as described elsewhere (29). NMAb 2G12 was not washed away
but rather was diluted 1:1 with fresh medium daily, starting on day 3 of the
experiment. Due to this assay condition, which takes into account the long
half-lives of antibodies, neutralization titers may differ slightly from titers mea-
sured by other methods (6, 36). Antibody-mediated neutralization is expressed as
a percentage of neutralization of virus infectivity (29); antibody concentrations
during the preincubation of 2G12 with virus ranged from 0.32 ?g/ml to 40 ?g/ml.
Lymphocyte immunophenotyping. PBMC isolated from Indian-origin rhesus
monkeys were stained for flow cytometric analysis using combinations of the
following fluorochrome-conjugated MAbs: anti-CD3-Alexa Fluor 700 (SP34-2;
BD Biosciences, San Jose, CA), anti-CD4-PerCP-Cy5.5 (L200; BD Biosciences),
anti-CD28-PE (28.2; BD Biosciences), and anti-CD95-PE (DX2; BD Bio-
sciences). The samples were analyzed by four-color flow cytometry (FACSCalibur;
BD Biosciences Immunocytometry Systems). Data analysis was performed by
using FLOWJO (TreeStar, San Carlos, CA). CD3?CD4?T cells were gated
based on CD28 and CD95 expression to define memory CD4?T-cell subpopu-
lations: naive (CD28?CD95?), central memory (CD28?CD95?), and effector
Nucleotide sequence accession number. The nucleotide sequence determined
in the course of this work was deposited in GenBank under accession number
SHIV-1157i infection and disease progression in rhesus
monkey RPn-8. We modified the SHIV-vpu? backbone to
express env of a relatively recently transmitted pediatric HIV
isolate (HIV1157i) from a 6-month-old Zambian infant born
8730 SONG ET AL.J. VIROL.
to an HIV-positive mother. SHIV-1157i contains most of
gp120 as well as the entire extracellular domain and trans-
membrane region of gp41 of HIV1157i (Fig. 1A). An infant
macaque, RPn-8, was inoculated intravenously with 6 ml of
SHIV-1157i stock. Persistent viremia (Fig. 2A) and signs of
pathogenicity were observed in this monkey. The memory
CD4?CD29?T-cell subset (Fig. 2B) (4, 51) as well as absolute
CD4?T cells (Fig. 2C) in this monkey became depleted, with
absolute CD4?T-cell counts below 200 cells/?l for more than
1 year. By the above criterion, this monkey developed AIDS
at week 137 postinoculation. Monkey RPn-8 also developed
thrombocytopenia at week 46 postinoculation.
Passage of uncloned, late-stage virus. To test whether more
aggressive progeny viruses had emerged during the protracted
chronic infection and gradual progression to AIDS in monkey
RPn-8, we transferred 10 ml of blood containing the late-stage
virus (SHIV-1157ipd) from RPn-8 intravenously to another
naı ¨ve rhesus monkey, RBg-9. Compared to the early-stage
virus SHIV-1157i, SHIV-1157ipd induced peak viremia (week
1 postinoculation) that was ?2 logs higher (Fig. 2D). RBg-9
developed persistent thrombocytopenia (data not shown) and
became relatively depleted of its CD4?CD29?memory T-cell
subset (Fig. 2E) 12 weeks after transfer of late-stage virus.
Isolation and characterization of late-stage virus. Our re-
sults in recipient animal RBg-9 are in agreement with previous
observations that SIV variants emerging during late-stage dis-
ease have a higher replicative capacity and increased pathoge-
nicity (28). Therefore, in an effort to obtain a more virulent
SHIV clade C strain, uncloned virus (SHIV-1157ipd) was re-
isolated from animal RPn-8 4 weeks after the onset of AIDS.
Genetic analysis of SHIV-1157ipd env sequences exhibited a
number of changes resulting in single-amino-acid substitutions
in gp120 (especially in V1, V2, and V4) and gp41. In addition,
a 118-bp deletion in the 3? end of gp41 was seen in all five
clones that were sequenced. This deletion led to a loss of the
last 35 amino acids in the original HIV-1 gp41 and a gain of the
last 57 amino acids of the SIVmac239 gp41 through frameshift
(Fig. 1C) relative to the parental SHIV-1157i provirus. These
changes are similar to, yet distinct from, the env changes that
occurred in SHIV89.6P during its serial passage (47). These
changes in gp41 represent a striking example of convergent
molecular evolution of different SHIV constructs in indepen-
dently inoculated rhesus monkeys. Compared with the paren-
tal SHIV-1157i, nef sequences in SHIV-1157ipd exhibited six
changes resulting in single-amino-acid substitutions (Fig. 1D).
We next determined coreceptor usage of late-stage virus.
SHIV-1157ipd replicated neither in CEMx174-GFP cells nor
in U87.CD4, U87.CD4.CCR1, U87.CD4.CCR2, U87.CD4.CCR3,
U87.CD4.CXCR4, GHOST-BOB, and GHOST-BONZO cells.
Productive infection occurred only in U87.CD4.CCR5 cells
(data not shown), indicating that SHIV-1157ipd exclusively
uses CCR5 as its coreceptor.
Construction and characterization of SHIV-1157ipd3. A
molecular clone of SHIV-1157ipd, termed SHIV-1157ipd3,
was constructed using SHIV-vpu? as the backbone and con-
taining the entire 3? half, including env and nef, of SHIV-
1157ipd. Genomic DNA from RPn-8 PBMC collected at week
141 after virus exposure (4 weeks after AIDS was diagnosed)
was used to amplify the SHIV-1157ipd 3? half, which was
ligated with the 5? half of SHIV-vpu? (31) to form the full-
length proviral DNA of SHIV-1157ipd clones (Fig. 1B). A total
of 23 full-length clones were produced and screened for their
infectivity on TZM-bl using the supernatant of transfected
293T cells. SHIV-1157ipd3 was picked due to its highest infec-
tivity among these clones. SHIV-1157ipd3 replicated well in
the PBMC of all six randomly selected naı ¨ve macaques (data
FIG. 1. Schematic representation of SHIV-1157ipd3 construction.
(A) Structure of SHIV-1157i. SHIV-1157i contains env of a relatively
recently transmitted pediatric HIV isolate from a 6-month-old Zam-
bian infant. A unique restriction site, PuvI (P), was introduced into the
3? half of SHIV-vpu? proviral DNA. The 2.0-kb KpnI (K)-PvuI frag-
ment of HIV1157i (spanning most of gp120 as well as the entire gp41
extracellular domain and the transmembrane region [TM]) was ampli-
fied to replace the corresponding region of SHIV-vpu? env. The
modified 3? half was ligated with the 5? half of SHIV-vpu? proviral
DNA to form full-length SHIV-1157i. (B) Construction of SHIV-
1157ipd3. The entire 3? half of SHIV-1157ipd3 (SphI-NotI fragment)
was amplified from the genomic DNA of PBMC isolated 4 weeks after
animal RPn-8 developed AIDS, as defined by persistent CD4?T-cell
counts of ?200 cells/?l. This fragment was ligated with the 5? half of
SHIV-vpu?, which was previously cloned into a modified pSP-73 vec-
tor, to form proviral DNA SHIV-1157ipd3. (C) Predicted amino acid
sequences of the C-terminal domains of gp41 in SHIV-1157i and
SHIV-1157ipd. (D) Predicted amino acid sequences of Nef in SHIV-
1157i and SHIV-1157ipd.
VOL. 80, 2006MUCOSALLY TRANSMISSIBLE R5 SHIV CLADE C8731
SHIV-1157ipd3 lacks the 2G12 epitope contained in
HIV1157i. The carbohydrate-dependent epitope of the human
NMAb 2G12 includes N-linked mannan moieties associated
with the five residues N295, N332, N339, N386, and N392. The
core epitope consists of glycans attached to N295, N332, and
N392 (52, 53). HIV1157i and SHIV-1157i have all five of these
N-linked glycosylation sites, but after almost 3 years of repli-
cation in monkey RPn-8, N295 was mutated to T295 in SHIV-
1157ipd3 (Fig. 3A). The other four asparagines associated with
the 2G12 epitope were retained in SHIV-1157ipd3. When we
evaluated the susceptibility of HIV1157i and SHIV-1157ipd3
to neutralization by human NMAb 2G12 in vitro, infection of
human PBMC by HIV1157i was inhibited by 2G12 in a dose-
dependent manner (Fig. 3B). However, SHIV-1157ipd3 was
not sensitive to 2G12 (Fig. 3C).
Increase in the virulence of SHIV-1157ipd3 by introduction
of an additional NF-?B binding site. One to three NF-?B
binding sites may be present in the enhancer regions of SIV or
different HIV-1 subtypes (9, 38, 46). We hypothesized that the
number of such sites in the viral LTR may affect viral replica-
tion and virulence. For instance, SIVmac239, the backbone
normally used for the construction of SHIV chimeras, has one
NF-?B binding element. However, duplication of NF-?B bind-
ing sites in monkey-passaged SIVs can result in acutely lethal
variants such as SIVpbj14 (17) or pathogenic progeny that had
evolved from live attenuated SIVmac239?3 (1). In the latter
case, progeny virus containing two NF-?B sites had replaced
the original virus, implying better in vivo replicative capacity of
virus that had emerged late in the course of the infection (1).
In addition, other investigators have described a direct corre-
lation between the number of NF-?B sites and LTR-driven
gene expression (26).
To directly test our hypothesis, we first evaluated a set of
solo-LTR constructs. The pLuc reporter plasmid with a trun-
cated portion of the SIVmac239 LTR (?2253?149; one
NF-?B binding site) upstream of the firefly luciferase reporter
gene was engineered by site-directed mutagenesis to produce
a total of three constructs containing different numbers of
NF-?B binding sites (Fig. 4A). Mutants ?2-4 and 138-4 contain
zero and two NF-?B binding sites, respectively, in their LTR
FIG. 2. Plasma viremia and CD4?T-cell loss due to SHIV-1157i (monkey RPn-8) and SHIV-1157ipd (monkey RBg-9). Infant macaque RPn-8
was inoculated intravenously with 6 ml of early-stage SHIV-1157i stock (left panels). Rhesus monkey RBg-9 received 10 ml of blood intravenously,
transferred from monkey RPn-8 4 weeks after the latter had CD4?T-cell counts of ?200 cells/?l; the transferred blood contained the late-stage
virus, SHIV-1157ipd (right panels). (A and D) Plasma viral RNA loads. The dashed lines indicates the lower level of sensitivity of the RT-PCR
assay (50 copies/ml) (24). (B and E) CD4?memory T-cell counts (CD4?CD29?). The dashed lines denote the lowest normal value (10%) for the
percent CD4?CD29?cells. (C and F) Absolute CD4?T-cell counts. The dashed lines denote 200 CD4?T cells. By definition, a persistent
peripheral CD4?T-cell count of ?200 cells/?l is indicative of AIDS. The arrow denotes the time of blood transfer of SHIV-1157ipd to animal
RBg-9 and also the time of reisolation of the SHIV-1157ipd biological isolate.
8732SONG ET AL. J. VIROL.
regions. Luciferase activity was measured in 293T cells tran-
siently transfected with one of the three mutant constructs with
or without cotransfecting a plasmid containing SHIV89.6 tat.
The transfected cells were also cultured in the presence or
absence of TNF-?, a known inducer of NF-?B activation, to
determine the level of transcription driven by each of the
mutant LTRs (Fig. 4B). The level of luciferase activity pro-
duced in 293T cells transfected with each of the solo-LTR
constructs was associated with the number of NF-?B binding
sites in the LTR enhancer region. The differences were most
pronounced in the presence of Tat and/or TNF-?. The highest
luciferase activity was observed in 293T cells transfected with
the mutant that has two NF-?B binding sites, 138-4, in the
presence of Tat and TNF-?. These results suggest that a viral
LTR with two NF-?B binding sites may be more responsive to
Tat and TNF-? than LTRs with zero or only one NF-?B
We next sought to test this concept in the context of repli-
cation-competent, isogenic viruses and to evaluate whether this
strategy could be used to enhance the replicative capacity of
SHIV-1157ipd3. To this end, a modified molecular clone,
SHIV-1157ipd3N4, was constructed by introducing an addi-
tional NF-?B binding site into the 3? LTR of SHIV-1157ipd3
by site-directed mutagenesis. We evaluated the growth of
SHIV-1157ipd3 and SHIV-1157ipd3N4 in PBMC from ran-
domly selected rhesus monkey donors in the presence or ab-
sence of TNF-?. Although both viruses replicated well in
PBMC from a naive donor (Fig. 4C), the highest peak p27
concentration was observed with SHIV-1157ipd3N4 in the
presence of TNF-?, suggesting that the latter virus may have a
replicative advantage over SHIV-1157ipd3 due to its extra
NF-?B binding site. The same pattern was observed in PBMC
from all three macaque donors tested (data not shown).
Coreceptor usage of late-stage SHIV strains. We next
assessed coreceptor usage of SHIV-1157ipd3 and SHIV-
1157ipd3N4. Neither virus replicated in any cell line lacking
CCR5, including CEMx174-GFP, U87.CD4, U87.CD4.CCR1,
U87.CD4.CCR2, U87.CD4.CCR3, U87.CD4.CXCR4, GHOST-
BOB, and GHOST-BONZO cells (Fig. 4E and data not shown).
The observation that productive infection occurred only in
U87.CD4.CCR5 cells (Fig. 4D) suggests that late-stage SHIV-
1157ipd3 and SHIV-1157ipd3N4, like the uncloned virus SHIV-
1157ipd, exclusively use CCR5 as their coreceptor.
Comparison of the infectivity of SHIV-1157ipd3N4 in rhesus
monkeys of Chinese or Indian origin. A large stock of SHIV-
1157ipd3N4 was generated in Indian-origin rhesus monkey
PBMC. Chinese rhesus monkeys were inoculated intrarectally
with various dilutions of the viral stock to determine the in vivo
infectivity. Undiluted stock and dilutions of 1:5.5 and 1:10
resulted in systemic infection of each of the exposed animals,
whereas a dilution of 1:50 led to infection in two out of three
animals. Dilutions of 1:60 or higher did not yield systemic
infection. Among the five monkeys that became systemically
infected, no correlation between the viral inocula and peak
viral RNA loads was seen, consistent with earlier observations
(11). The monkey with the highest peak viral RNA load of
8.2 ? 106copies/ml had received 1 ml of a 1:50 dilution of the
original stock (Fig. 5A). The statistical method of Spouge (55)
was used to determine the 50% animal infectious dose of the
SHIV-1157ipd3N4 stock for the intrarectal route in Chinese-
origin rhesus monkeys; the value was 2.46 ? 10?2ml. During
the 40-week follow-up period, three out of the five Chinese
rhesus monkeys have maintained high viral loads (Fig. 5A).
We next assessed intrarectal transmission in eight Indian
rhesus monkeys (Fig. 5B). These animals were inoculated in-
trarectally with 0.5 ml of the same large-scale SHIV-
1157ipd3N4 stock and monitored for infection. All eight ma-
caques showed robust viral replication during the first 2 weeks
postinoculation, with peak viral RNA loads ranging from 1.4 ?
107to 7.1 ? 107copies/ml of plasma. The average peak viral
RNA load of SHIV-1157ipd3N4 in the eight Indian-origin
rhesus monkeys was 11 times higher than that in the five
Chinese-origin rhesus monkeys (two-sided P ? 0.002; Wil-
coxon rank-sum test). During the 29-week follow-up period,
three out of the eight Indian rhesus monkeys have maintained
FIG. 3. Neutralization of HIV1157i and SHIV-1157ipd3 with neu-
tralizing monoclonal antibody 2G12. (A) Amino acid sequence align-
ment of the 2G12 epitope. The boxed amino acid residues show the
critical mutation at position 295. (B) Neutralization of HIV1157i by
NMAb 2G12. (C) Neutralization of SHIV-1157ipd3 by 2G12. Neutral-
ization assays were performed in phytohemagglutinin (PHA)-activated
human PBMC in triplicate. Virus was incubated with the NMAb 2G12
or an isotype control NMAb at the indicated concentrations for 1 h at
37°C and then added to PBMC with IL-2; NMAbs were not washed
out. Virus production was assessed by measuring p24 or p27 levels in
the culture supernatants. IgG, immunoglobulin G.
VOL. 80, 2006 MUCOSALLY TRANSMISSIBLE R5 SHIV CLADE C8733
FIG. 4. Number of NF-?B sites and LTR-mediated gene expression, replication kinetics of SHIV-1157ipd3 and SHIV-1157ipd3N4, and
coreceptor usage of the late viruses SHIV-1157ipd3 and SHIV-1157ipd3N4. (A) Schematic representation of pLuc mutant constructs containing
various numbers of NF-?B sites. The pLuc reporter plasmid with a truncated portion of the SIVmac239 LTR upstream of the firefly luciferase
reporter gene was engineered to yield three constructs with different numbers of NF-?B binding sites. Isolates ?2-4, wild-type (wt), and 138-4
contain zero, one, and two NF-?B binding sites, respectively, in their LTR enhancer regions. (B) Luciferase expression of each of the constructs
in transfected 293T cells with or without TNF-? or a plasmid containing SHIV89.6 tat (pTat). Luciferase activity was measured to determine the
level of gene expression driven by each LTR. The amount of luciferase activity produced in 293T cells transfected with the indicated plasmid was
divided by that of 293T cells transfected with wt plasmid only (without TNF-? or pTat) to calculate fold induction. Error bars represent standard
deviations of the experiments. (C) PBMC (2 ? 106) from a randomly selected rhesus monkey (CF-37) were activated with ConA and exposed to
either SHIV-1157ipd3 (one NF-?B site per LTR) or SHIV-1157ipd3N4 (two NF-?B sites per LTR) produced from transiently transfected 293T
cells, each at 30 ng of p27 Gag protein in the presence (closed symbols) or absence (open symbols) of human TNF-? (10 ng/ml). Levels of p27
were measured in the PBMC supernatants on the indicated days following virus exposure. U87.CD4.CCR5 cells (D) and U87.CD4.CXCR4 cells
(E) were exposed to SHIV-vpu?, SHIV-1157ipd3, SHIV-1157ipd3N4, or SHIVSF162P3. Supernatants were harvested and replaced daily. Levels of
p27 Gag were measured in the supernatants on the indicated days following virus exposure.
8734 SONG ET AL. J. VIROL.
high viral loads. Five more Indian rhesus monkeys were also
inoculated intrarectally with various dilutions of the SHIV-
1157ipd3N4 stock. Dilutions of 1:10, 1:50, and 1:60 resulted in
systemic infection of the exposed animals, whereas a dilution
of 1:100 led to infection in one out of two animals (unpublished
data). These data indicate that intrarectal transmission of our
SHIV-1157ipd3N4 stock is reproducible.
We then estimated the replicative capacity of SHIV-
1157ipd3N4, the late virus, compared to that of SHIV-1157ip,
the early virus, after mucosal inoculation of Indian-origin
rhesus macaques. Mean peak viral RNA load in SHIV-
1157ipd3N4-infected animals (n ? 8) was 4.05 ? 107copies/ml,
compared to 9.90 ? 106viral RNA copies/ml in SHIV-1157ip-
infected animals (n ? 19; P ? 0.000284; Student’s t test). These
results indicate that SHIV-1157ipd3N4 has a significantly
higher replicative capacity than SHIV-1157ip.
Early signs of pathogenicity of SHIV-1157ipd or SHIV-
1157ipd3N4. The uncloned late virus SHIV-1157ipd, which
was transferred to juvenile rhesus macaque RBg-9 by intrave-
nous blood transfer from RPn-8, induced memory CD4?
CD29?T-cell depletion (Fig. 2E) and thrombocytopenia (data
not shown) in the new host within 3 months of inoculation.
Among Indian-origin rhesus macaques inoculated with SHIV-
1157ipd3N4, a sharp reduction in CD4?central memory T
cells (CD28?CD95?) was observed in two animals (monkeys
RPo-10 and RCp-10) out of the five animals tested (Table 1).
The central memory T-cell population in animal RCp-10
dropped from 17% at week 0 to 5.8% at week 8. However, in
all eight Indian-origin and five Chinese-origin animals, abso-
lute peripheral CD4?T-cell counts remained within the nor-
mal range during the early period of observation (data not
shown). Longer term pathogenicity studies are needed to as-
sess the rate of progression to immunodeficiency for these
late-stage virus strains.
The overall goal of this study was to generate a highly rele-
vant R5 SHIV that encodes HIV-1 clade C env. To this end, we
have generated a series of related SHIVs that carry env of a
pediatric HIV-1 clade C strain. The early SHIV forms, SHIV-
1157i and SHIV-1157ip, are exclusively R5 tropic and induced
progressive disease after 2.6 to almost 5 years in three out of
the five rhesus monkeys used in the initial virus adaptation.
While this rate of disease progression is consistent with lenti-
FIG. 5. Intrarectal inoculation of SHIV-1157ipd3N4. Five Chinese-origin rhesus monkeys (A) and eight Indian-origin rhesus monkeys (B) were
inoculated intrarectally with SHIV-1157ipd3N4 stock with concentrations noted in the text. Viral loads were measured at indicated time points
postinoculation. The dashed lines indicate the lower level of sensitivity of the RT-PCR assay (50 copies/ml).
TABLE 1. T-cell populations of Indian-origin rhesus monkeys inoculated with SHIV-1157ipd3N4
T-cell population at week:
aEM, effector memory T cells.
bCM, central memory T cells.
cValues in boldface are abnormal.
VOL. 80, 2006MUCOSALLY TRANSMISSIBLE R5 SHIV CLADE C 8735
viral biology, faster disease progression rates would render the
SHIV clade C/rhesus monkey model more practical. To gen-
erate more virulent versions of the virus, we employed a dual
approach: first, we reisolated late-stage virus from the first
monkey that had progressed to AIDS, and second, we engi-
neered an extra NF-?B site into the LTR of the molecularly
cloned late-stage virus to boost its replicative capacity and
responsiveness to TNF-?. The resulting infectious molecular
clone, SHIV-1157ipd3N4, has a number of biologically rele-
vant characteristics: (i) it is exclusively R5 tropic; (ii) it is
mucosally transmissible in rhesus macaques; and (iii) infection
with SHIV-1157ipd3N4 induces high peak viral RNA loads in
all animals tested and high viral set points in 10 out of 13
infected animals. Although SHIV-1157ipd3N4 lacks the acute
pathogenicity of typical X4 or X4R5 SHIVs, it induced abnor-
malities in immune parameters, such as central memory CD4?
T-cell depletion and decreased CD4/CD8 ratios, during a rel-
atively short period of postexposure followup.
SHIV-1157ipd3N4 carries the envelope gene of the HIV
clade C, the most prevalent substrain worldwide. Although
four SHIV strains (SHIVCHN19, SHIVMJ4, SHIV-MCGP1.3,
and SHIV-XJ02170) encoding clade C envelopes have been
created thus far, they either are dual tropic (SHIV-MCGP1.3)
(8), are unable to replicate in rhesus macaque PBMC
(SHIVCHN19) (10), or show no evidence thus far of mucosal
XJ02170) (41, 58). In contrast, SHIV-1157ipd3N4 was muco-
sally transmissible and exhibited uniform robust viral replica-
tion kinetics during acute viremia; in fact, a high-titer stock has
been titrated by the intrarectal route. Thus, this new R5 clade
C SHIV can be used to assess vaccine efficacy, with prevention
of infection, lowering of peak viral loads, and lowering of
postacute viremia levels as read-out parameters. Longer term
follow-up studies of SHIV-1157ipd3N4-infected monkeys will
determine whether protection from disease progression will be
an additional criterion.
Genetic analysis of our data showed a major deletion in the
cytoplasmic tail of the SHIV-1157ipd env gene that ended
immediately proximal to the start codon of nef compared with
the parental SHIV-1157i. This deletion led to the removal of
the original stop codon of HIV-1 env and the overlapping of
env and nef in the resultant SHIV-1157ipd. Therefore, the new
cytoplasmic tail of gp41 contains both HIV-1 and SIVmac239
sequences, similar to the pattern of the cytoplasmic tail of
SHIV89.6P env (47). This suggests that the carboxy-terminal
cytoplasmic tail of SIVmac239 gp41 plays a very important role
in the pathogenesis of SHIVs in rhesus monkeys.
The exclusive R5 tropism of SHIV-1157ipd3N4 represents a
more biologically relevant coreceptor usage for mucosally
transmitted viruses than the dual tropism of SHIV89.6P (47,
49), which has been widely used as the challenge virus in
macaque vaccine trials (3, 5, 54). It is noteworthy that SHIV-
1157ipd3N4 infection does not induce the acute, severe patho-
genicity typically seen within 2 weeks of SHIV89.6P infection,
which does not reflect the biology of acute HIV-1 infection in
humans. Of note, SHIV89.6P infects and destroys naı ¨ve CD4?
T cells, whereas typical R5 viruses, such as SIVmac239 and
SIVsmE543, affect predominantly the central memory T-cell
subset (43). Indeed, the biological relevance of the SHIV89.6P
challenge model has been called into question for these rea-
sons (20, 42, 43). In contrast to SHIV89.6P, the gradual patho-
genicity of SHIV-1157i and SHIV-1157ip (early form) more
closely reflects the pattern of HIV-1 disease progression in
humans, which is characterized by years of clinically stable,
chronic infection before immune exhaustion sets in. While we
have ruled out acute, severe pathogenicity of the late forms
SHIV-1157ipd as well as SHIV-1157ipd3N4, the currently on-
going prospective studies will reveal the rates of disease pro-
gression in rhesus monkeys.
SHIV-1157ipd3N4 is the first highly replication-competent,
mucosally transmissible R5 clade C SHIV. The fact that it
induces reproducibly high peak viral RNA loads with high viral
set points will allow viremia to be used as a readout in vaccine
challenge studies (Rasmussen et al., unpublished).
Our late-stage virus, SHIV-1157ipd3N4, models another as-
pect of most HIV-1 clade C strains: it has an extra NF-?B site
in the LTR compared to the standard version of the viral LTR
(as in SIVmac239). Most HIV-1 clade B isolates are charac-
terized by two NF-?B binding sites, whereas the majority of
HIV-1 clade C isolates have an extra NF-?B binding site (23,
39). The number of NF-?B binding elements is associated with
the efficacy of transcriptional initiation from the proviral ge-
nome, and TNF-? leads to the up-regulation of NF-?B (19,
44). In addition, a correlation between HIV-1 LTR subtype
configuration of the NF-?B promoter region and responsive-
ness to TNF-? was reported earlier (37). The data derived
from the in vitro pLuc expression controlled by a truncated
LTR with zero to two NF-?B binding elements support the
previously held notion (26, 37, 40) that the number of NF-?B
binding sites in the LTR directly correlates with the level of
gene expression driven by the LTRs. As expected, the addi-
tional NF-?B binding element in the SHIV-1157ipd3N4 LTRs
rendered our virus more responsive to the effects of TNF-?
In our intrarectal transmission studies of SHIV-1157ipd3N4,
significantly higher peak viral RNA loads (week 2) were ob-
served in Indian-origin rhesus macaques than in Chinese-ori-
gin rhesus macaques (Fig. 5A and B). Since we reisolated our
late-stage virus after it replicated for 141 weeks in the Indian-
origin monkey RPn-8, which had been given the original con-
struct, we postulate that the late-stage virus was better adapted
and more replication competent in Indian-origin monkeys
than in Chinese-origin monkeys. Although some Chinese-ori-
gin rhesus monkeys received lower viral inocula, we and others
have demonstrated a lack of any correlation between inoculum
size, peak viremia levels (11), viral set points, and disease
progression (7). This observation was made not only with in-
trarectal inoculation but also with oral administration (11).
This lack of a correlation is not surprising for outbred popu-
lations of primates and is consistent with the idea that systemic
infection occurs once the mucosal virus inoculum exceeds a
minimal dose (11). Although previous studies have shown that
the set points of SIV or SHIV in Indian-origin rhesus monkeys
are generally higher than in Chinese-origin rhesus monkeys,
conflicting results on the peak viremia have been observed (32,
33, 48, 56). Our data showed that the peak viremia of SHIV-
1157ipd3N4 was significantly higher in Indian-origin rhesus
monkeys than in Chinese-origin rhesus monkeys. Despite the
differences in viral parameters postinoculation, our data show
8736SONG ET AL. J. VIROL.
that SHIV-1157ipd3N4 can be used for intrarectal challenges
in rhesus macaques regardless of their origin.
Our in vivo data indicate that SHIV-1157ipd3N4 is a highly
replication-competent R5 clade C SHIV that can reproducibly
infect rhesus macaques mucosally. Because its coreceptor us-
age reflects the tropism of the mucosally transmitted forms of
HIV-1, SHIV-1157ipd3N4 may represent a practical tool to
test the efficacy of vaccine candidates targeting Env of the
world’s most prevalent clade, HIV clade C, in a relevant pri-
We thank Joseph Sodroski (Dana-Farber Cancer Institute, Boston,
MA) for the gift of the SHIV-vpu? proviral clones, Hermann Katinger
(Polymune Scientific, Vienna, Austria) for NMAb 2G12, K. A. Buckley
for technical support, Susan Sharp for assistance in the preparation of
the manuscript, Daniel Anderson for pathology support, Stephanie
Ehnert for coordinating sample collections, and Patrick Autissier for
technical support in fluorescence-activated cell sorter analysis.
This work was supported in part by NIH grants R01 DE12937, R01
DE0160354, and R37 AI34266 to R.M.R., HD39620 and RR15635 to
C.W., P01 AI48240 to R.M.R., C.W., and H.M.M., and RR00165,
which provided base grant support to the Yerkes National Primate
Research Center. J.B.W. was supported by a postdoctoral fellowship
from the Canadian Institutes for Health Research.
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