Variable region 4 of SIVenvelope correlates with rapid disease progression in
morphine-exposed macaques infected with SIV/SHIV
Vanessa Rivera-Amilla,c,⁎, Richard J. Noel Jr.a,b, Suheydi Orsinid, Griselle Tiradoa,d,
José M. Garcíad, Shilpa Buche, Anil Kumara,c,d,f
aAIDS Research Program, Ponce School of Medicine, Ponce, PR 00732, Puerto Rico
bDepartment of Biochemistry, Ponce School of Medicine, Ponce, PR 00732, Puerto Rico
cDepartment of Microbiology, Ponce School of Medicine, Ponce, PR 00732, Puerto Rico
dLaboratory of Viral Immunology, Ponce School of Medicine, Ponce PR 00732, Puerto Rico
eDepartment of Physiology, KU Medical Center, Kansas City, KS 66160, USA
fDepartment of Pharmacology, School of Pharmacy, University of Missouri, Kansas City, KS 64108, USA
Received 20 June 2006; returned to author for revision 1 August 2006; accepted 23 August 2006
Available online 28 September 2006
We analyzed the association between the evolution of the V3–V5 regions of env and disease progression in our SIV/SHIV macaque model of
morphine dependence and AIDS. Previous studies revealed two distinct disease patterns—fast progression and normal progression. To determine
the effect of the two distinct patterns of disease in the evolution of SIV/17E-Fr envelope, we analyzed env sequences from three morphine-
dependent macaques that developed accelerated AIDS and three morphine-dependent macaques that developed AIDS at a slower rate and
compared them to control macaques. Morphine-dependent animals exhibited a higher percentage of diversity in both plasma and CSF
compartments within V4 when compared to controls. Divergence from the inoculum was significantly greater in the morphine group as compared
to controls in CSF but not in plasma. We also found a direct correlation in V4 evolution and rapid disease progression. These results indicate that
morphine dependence plays a role in the pathogenesis of SIV/SHIV infection and env evolution.
© 2006 Elsevier Inc. All rights reserved.
Keywords: SIV; SHIV; Envelope evolution; Macaque; Morphine; Variable region
Injection drug use (IDU) continues to be an important cause
of human immunodeficiency virus (HIV) infection in the United
States and other countries (CDC, 2003; Chu and Levy, 2005;
Kerr, 2005; Qian et al., 2006; Cohn, 2002). The relationship
between IDU and the differences in the virology and
immunopathogenesis of HIV and its effect in antiretroviral
treatments remains to be elucidated. Human studies on the
influence of IDU in disease progression and survival rates
although meaningful, provide insufficient information because
of the many factors present among injection drug users
unrelated to HIV. However, there are ample indirect proofs
that illicit drug abuse may adversely affect HIV/AIDS. These
include (i) morphine-mediated increase in HIV-1 replication in
tissue culture (Ho et al., 2003; Li et al., 2003; Guo et al., 2002;
Schweitzer et al., 1991); (ii) AIDS as major cause of death in
IDU cohort (Tyndall et al., 2001; CDC, 2001); and (iii)
morphine-mediated exaggeration of bacterial infection, includ-
ing direct correlation between bacterial infections like tubercu-
losis and AIDS (Cohn, 2002).
In order to circumvent major gaps in HIV human studies,
various animal models of HIV/AIDS have been established,
allowing for a better control of parameters (Bonyhadi and
Kaneshima, 1997; Esparza, 1990; Fultz, 1993; Gardner, 1990;
Kinman et al., 2004; Persidsky et al., 1995, 2005; van Maanen
and Sutton, 2003; Vodros and Fenyo, 2004). These animal
models have provided a way to test pre-clinical effectiveness of
drugs, develop vaccines, examine virulence, and determine the
role of antiviral immune responses, among others. One such
Virology 358 (2007) 373–383
⁎Corresponding author. AIDS Research Program Ponce School of Medicine,
Ponce, PR 00732-7004, Puerto Rico. Fax: +1 787 841 5150.
E-mail address: firstname.lastname@example.org (V. Rivera-Amill).
0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
model, the non-human primate has proven to be particularly
valuable for the study of viral pathogenesis and disease prog-
ression because of the similarities between simian immunode-
ficiency virus (SIV) AIDS-like illness and HIV-AIDS
(Haigwood, 2004; Hu, 2005; Levy, 1996; Vodros and Fenyo,
In view of the fact that both survival advantage and dis-
advantage have been reported in the setting of drug abuse and
that results from animal studies are inconclusive, we developed
a non-human primate model of drug abuse and AIDS in Indian
rhesus macaques. In our model, we use a mixture of three
viruses that had been previously shown to cause massive CD4+
cell loss and neurological disorders in animals (Kumar et al.,
2004a, 2006). Using this model, we have previously shown that
morphine-dependent macaques showed significantly higher
virus replication. We have also demonstrated that 50% of the
morphine-dependent and infected animals develop SHIV/SIV-
induced disease within 20 weeks after infection whereas other
morphine-dependent and control animals survived for much
In our attempt to establish a reason for accelerated disease
progression in half of the morphine-dependent macaques, we
sought to determine whether there was a correlation between
virus evolution and disease progression. Earlier studies from
this laboratory have indicated an inverse correlation between
SIVtat evolution both in plasma as well as cerebrospinal fluid
(CSF) compartments (Noel and Kumar, 2006; Noel et al.,
2006). This observation was later extended to our study on SIV
envelope (env) wherein we found similar correlation within
variable regions 1 and 2 (Tirado and Kumar, 2006). In this
study, we sought to characterize the changes within env V3–V5
regions to determine whether there is an indirect correlation as
was seen previously, or if evolution of some variable regions of
env is directly related to disease progression in morphine-
dependent macaques. The data presented represent a compre-
hensive analysis of envelope genetic variation in plasma and
CSF from rapid and normal progressor morphine-dependent
and control macaques. Our results indicate a direct correlation
between env V4 evolution and rapid disease progression in
We have sequenced and analyzed a total of 125 plasma and
123 CSF SIV envelope (env) clones, covering the variable
regions V3–V5 from six morphine-dependent (1/04L, 1/28Q, 1/
42N, 1/02N, 1/52N, and 1/56L) and three control rhesus
macaques (2/02P, 2/31P, and 2/AC42) to assess compartmenta-
lization of viral evolution. Plasma and CSF were collected at
regular intervals from each macaque after experimental
inoculation with SIV/SHIV. An earlier study showed that mor-
macaques (Kumar et al., 2004a). Among morphine-dependent
macaques, half progressed rapidly, maintained high viral loads
and low CD4+T-cell counts (1/04L, 1/28Q, and 1/42N). These
animals, termed rapid progressors, died on or before the 20th
week post-infection. The other three animals within the
intermediate viral loads and CD4+T-cell counts (1/02N, 1/52N,
and 1/56L). The control animals showed the least severe disease
as compared to the rapid progressors and moderately better than
the normal progressors (Noel and Kumar, 2006; Noel et al.,
2006; Kumar et al., 2004a, 2006). Retroviral infections are
characterized by different levels of viral genetic variation within
as replication, mutation, and recombination and by host factors
such as immune response and target cell range (van Marle and
Power, 2005; Negroni and Buc, 2001; Nowak, 1992). In this
dependent macaques and compared it to control group to
elucidate whether morphine exerts any effect over viral
evolution. We also analyzed the rapid progressors and normal
progressors from the morphine-dependent group and compared
them to control macaques to determine any correlation between
pressure as evidenced by the changes seen in the rapid
progressors which we had previously shown to lack effective
humoral and cellular immune responses (Kumar et al., 2006).
Contrasting env diversity in plasma and CSF
Sequence data were obtained from clones derived from
collection at weeks 12 and 18 post-infection (wpi). Viral RNA
was extracted and amplified by RT-PCR prior to cloning and
sequencing. The sequenced clones represented a ∼905 base pair
fragment encompassing the V3 to V5 regions of SIV env.
Phylogenetic analysis of plasma and CSF-derived clones
within 18 wpi revealed differences in the evolution of the viral
quasi-species within the two compartments. Neighbor-joining
trees indicated that there is better compartmentalization in the
normal progressors and control macaques than in the rapid
progressors (Fig. 1). We then examined the sequences in each
animal independent of time to determine the overall diversity
and the nucleotide distance from the SIV/17E-Fr inoculum
(divergence). Analysis of the plasma-derived clones revealed a
significant difference (p=0.005) in the overall sequence
diversity of the clones from morphine-dependent macaques
(0.98%) when compared to control macaques (1.27%) with the
control macaques exhibiting a higher percentage (Fig. 2A).
There is no statistically significant difference in divergence for
the morphine group when compared to control group. Analysis
of the two morphine sub-groups revealed no significant
difference when comparing the diversity of the rapid
progressors to control macaques. However, a significant
difference (p=0.003) was detected when comparing the diver-
sity in the normal progressors (0.83%) to control macaques
(1.27%) (Fig. 2A). A statistically significant (p=0.010)
difference was also detected for the divergence from the
SIV/17E-Fr inoculum for the normal progressors (0.50%) but
not for the rapid progressors (0.80%) when compared to con-
trol macaques (0.73%) (Fig. 2B).
In CSF, as seen in plasma, there was a significant difference
(p=0.05) in the overall sequence diversity of the clones from
374 V. Rivera-Amill et al. / Virology 358 (2007) 373–383
morphine-dependent macaques when compared to control
macaques. However, in contrast to plasma, the morphine-
dependent macaques showed greater diversity than controls
(1.02% and 0.83%, respectively) (Fig. 2A). Analysis of the
divergence from the SIV/17E-Fr inoculum revealed that there
was a significant difference between the two groups (p=0.03),
with the morphine-dependent macaques exhibiting a higher
average percentage of divergence (0.68%) as compared to the
control macaques (0.50%) (Fig. 2B). Analysis of the morphine
sub-groups revealed a significant difference (p=0.03) in
diversity between the rapid progressors (0.97%) and control
macaques (0.83%) but no significant difference between normal
progressors and control macaques. Divergence from the SIV/
17E-Fr inoculum was only significantly different (p=0.05)
between the normal progressors (0.77%) and the control
macaques (0.50%) (Fig. 2B).
Analysis of the diversity and divergence of the clones
between plasma and CSF compartments revealed significant
differences in diversity in rapid progressors and control
macaques with the plasma-derived clones exhibiting higher
diversity, whereas the normal progressors exhibited a higher
divergence in the CSF compartment as compared to plasma
Evolution within individual variable regions shows conflicting
pattern of responses to morphine, compartment and rate of
Overall analysis of diversity and divergence within the
envelope variable regions in plasma-derived clones revealed an
inverse correlation in SIV env evolution between morphine-
dependent and control macaques within variable region 3. The
morphine-dependent macaques exhibited a 1.31% in diversity
and 0.94% in divergence whereas the control macaques
showed significantly greater (p=0.04) diversity (2.07%) and
divergence (2.07%) (Fig. 3A). No significant differences
where detected within variable region 3 from CSF-derived
In contrast, within variable region 4, a direct correlation in
diversity was detected for both plasma and CSF, in morphine-
dependent macaques when compared to controls. Morphine-
dependent plasma-derived clones exhibited a 1.48% diversity
vs. a 0.47% diversity in control macaques (p=0.04) (Fig. 3A).
Morphine-dependent CSF-derived clones exhibited a 1.75%
diversity vs. 0% diversity in control macaques (p=0.004) (Fig.
3B). Although the percentage of diversity in CSF was higher
than in plasma, statistical analysis revealed no significant
difference in diversity between the two compartments. In both
plasma and CSF, the rapid progressors exhibited a significantly
higher percentage in diversity when compared to control
macaques (Fig. 3).
Analysis of the overall plasma sequence diversity and diver-
gence of env within variable region 5 revealed no significant
differences in morphine-dependent animals when compared to
control macaques. However, a significant difference (p=0.02)
was detected in the divergence from the SIV/17 E-Fr inoculum
of the morphine-dependent CSF-derived clones when compared
to control macaques (1.19% and 0.33%, respectively) (Fig. 3B).
No significant differences in diversity and divergence within V5
were detected when comparing the two morphine sub-groups to
control macaques from both compartments.
Analysis of env variable region evolution at each time point
Plasma shows significant changes within V3 and V4
We also analyzed the nucleotide distance within and between
groups in the variable regions over time. As shown in Fig. 4,
plasma-derived clones from the morphine group exhibited a
was only significant at 18 wpi (morphine: 1.32%, control: 0.6%;
p=0.045). Comparison of the plasma-derived morphine sub-
groups revealed a significant difference when comparing the
rapid progressors to control but not when comparing the normal
progressors to control. The rapid progressors exhibited a 2.57%
in diversity at 12 wpi (p=0.009) and 2.2% at 18 wpi (p=0.010)
at 12 and 18 wpi, respectively. Analysis of the divergence from
the SIV/17E-Fr revealed a significant difference only when
comparing the rapid progressors to control group at 12 wpi
(rapid progressors: 2.17%, control: 0.23%; p=0.05).
No significant differences were detected in diversity over
time for V3 and V5. Divergence, however, within V3 was
significantly lower at 12 wpi in the morphine group (0.87%)
than in controls (1.93%, p=0.009). Among morphine-
dependent macaques, the rapid progressors were significantly
less divergent from the inoculum than the controls (0.75% vs.
1.93%, p=0.031) while the morphine-dependent normal pro-
gressors were less divergent (1.23%) but not at a statistically
divergence over time within V5 from plasma-derived clones
(data not shown).
CSF shows contrasting pattern with most significant changes
Analysis of the diversity of CSF-derived clones within
variable regions revealed that the morphine group exhibited a
higher percentage of diversity (12 wpi: 2.1%, p=0.003; 18 wpi:
6.2%, p=0.005) and divergence (12 wpi: 1.2%, p=0.002; 18
wpi: 1.1%) within variable region 4 at 12 and 18 weeks post-
infection when compared to control macaques (diversity and
diversity when comparing the rapid progressors to control at 12
1.07%, p=0.017; control: 12 and 18 wpi: 0%). Comparison of
the normal progressors to control also revealed a significant
difference but only at 12 wpi (normal progressors: 1.17%,
control: 0%; p=0.011). Between the morphine and control
diversity and divergence within variable region 3 (data not
shown). Within variable region 5, there is only a significant
difference in diversity between the normal progressors (1.4%)
and control group (0.73%, p=0.015) at 18 wpi. Analysis of
the divergence from the SIV/17 E-Fr inoculum revealed that
375V. Rivera-Amill et al. / Virology 358 (2007) 373–383
Fig. 1. Phylogenetic reconstruction of SIV env of plasma and CSF sequences from morphine-dependent and control macaques infected with SHIVKU-1B, SHIV89.6P,
and SIV/17E-Fr. DNA sequenceswere derived fromRT-PCRof viral RNA extractions fromplasma (solid symbols) and CSF (open symbols) at weeks12 (circles) and
18 (triangles) post-infection. The phylogenetic trees were generated by the neighbor-joining method using the MEGA 3.1 program. The trees were rooted using the
corresponding sequence from SIV/17E-Fr env (□). All trees are drawn to the same scale. The distance scale is indicated in the lower right of the figure.
376 V. Rivera-Amill et al. / Virology 358 (2007) 373–383
there is a significant difference between morphine and control
group at 18 wpi (morphine: 1.23%, control: 0.37%; p=0.033)
but not at 12 wpi. In both compartments, the rapid progressors
exhibited higher diversity in V4 when compared to control
Significant differences in amino acid changes within V4 are
detectable earlier in infection
Based on the deduced amino acid sequences, the majority of
amino acid changes occurred outside of the variable regions
(data not shown). Most of the amino acid changes within
variable regions 3 to 5 represent similar substitutions, indicating
a single evolutionary event.
Analysis of the overall frequency of amino acid mutations in
plasma-derived clones from morphine-dependent macaques
revealed a higher frequency of mutations in the morphine group
within V4 as compared to control macaques (0.619 and 0.99,
respectively; p=0.04). This difference is significant at 12 wpi
but not at 18 wpi (Table 1) indicating that mutations accumulate
in V4 early after infection. Comparison of the morphine sub-
groups with control macaques revealed a significant difference
in the rapid progressors within V4 (p=0.05) and V5 (p=0.008)
at 12 wpi but not at 18 wpi (Table 1). No statistically significant
frequency of amino acid mutations was detected either in V3 for
the rapid progressors or in V3 to V5 for the normal progressors.
As with plasma-derived clones, analysis of the overall
frequency of amino acid mutations in CSF-derived clones from
morphine and control groups revealed a higher frequency of
mutation in the morphine group within V4 (morphine: 0.611;
control: 0.260; p=0.003). However, over time this difference
was significant at 12 and 18 wpi (Table 1). In contrast to
plasma-derived clones, significant differences were also
detected for V3 and V5. Morphine-dependent CSF-derived
clones exhibited a higher frequency of amino acid mutations
within V5 (morphine: 0.410, control: 0.152, p=0.02) and this
difference was significant at 18 wpi but not at 12 wpi. Whereas,
the control group exhibited a higher frequency of amino acid
mutations within V3 (morphine: 0.050, control: 0.078; p=0.03)
and this difference was significant at 12 wpi but not at 18 wpi
(Table 1). Comparison of the morphine sub-groups with control
macaques revealed no significant differences between rapid
progressors and control macaques within V3 and significant
differences within V4 (p=0.004) and V5 (p=0.023) with the
rapid progressors exhibiting higher frequency of amino acid
changes, but only significant at 12wpi. The normal progressors
exhibited a significantly higher frequency of amino acid
mutations within V4 (0.514, p=0.003), and V5 (0.330,
p=0.044) when compared to control macaques (V4: 0 and
V5: 0.048), whereas as the control macaques exhibited a higher
frequency of mutation within V3 (0.147 vs. 0, p=0.019).
Injection drug use continues to be one of the major cofactors
among HIV positive individuals as more than 25% of persons
living with AIDS have been reported to belong to this category
in United States of America (2003). Several studies with this
cohort of HIV-infected individuals have failed to provide a
direct correlation between drug abuse and the impact on HIV
infection and disease progression because of the many factors
unrelated to HIV (Alcabes and Friedland, 1995).
Fig. 1 (continued).
377 V. Rivera-Amill et al. / Virology 358 (2007) 373–383
In HIV, evolution of env during the course of infection
allows for infection of a broader range of target cells and this
shift influences viral pathogenesis and disease course. These
changes may be a combinatorial result of viral replication/
recombination and the host's selective pressure. Regardless of
how env is modulated, the changes generated may affect
distinct domains of env, which have discrete functions in viral
pathogenesis. Among the regions of env, the V1–V2 region
and the V3 loop have been implicated as determinants in cell
tropism and co-receptor utilization, with the V3 playing a
major role in this function (Cho et al., 1998; Harrowe and
Cheng-Mayer, 1995; Hoffman et al., 1998). Studies within the
V4 region have revealed that it can tolerate substantial changes
in length, amino acid composition, and glycosylation without
compromising function (Ren et al., 2005). Thus, suggesting
that there is a minimal contribution of the V4 region to HIV-1
envelope glycoprotein function. However, because of its
exposure on the assembled envelope glycoprotein trimer and
its hypervariability, V4 may play a pivotal role in immune
evasion. In contrast, other studies have revealed a more active
role of the V4–V5 region in co-receptor utilization (Cho et al.,
1998; Hu et al., 2000; Smyth et al., 1998).
The SIV/SHIV morphine-dependent macaque model of
AIDS provides an ideal system in which to investigate the
molecular basis of antigenic variation because of the
similarities to human AIDS, and the capacity to control the
inoculum, timing, and drug exposure. In our model system, we
have seen that morphine accelerates the onset of disease.
Morphine-dependent macaques exhibited a more prominent
loss of CD4+T cells, higher viral set point, and more
pronounced viral replication in the cerebral compartment.
Thus, indicating positive correlation between morphine and
levels of viral replication (Kumar et al., 2004a, 2006). We
wanted to determine whether morphine could potentiate viral
evolution in the same way as disease progression. However,
studies on SIV tat evolution, both in plasma and CSF
Fig. 2. Diversity and divergence of env in plasma and CSF from morphine-dependent and control macaques. The env sequences in plasma (solid bars) and CSF (open
bars) from morphine-dependent and control macaques were aligned and used to calculate diversity (A) and divergence (B) using MEGA 3.1. The mean percent
diversity and divergence are plotted for each monkey. Error bars indicate mean of standard error. *P value of the comparison between plasma and CSF.
378 V. Rivera-Amill et al. / Virology 358 (2007) 373–383
compartments, revealed an inverse correlation with disease
progression (Noel and Kumar, 2006; Noel et al., 2006).
Morphine-dependent macaques exhibited a lower tat sequence
diversity and divergence in plasma (Noel and Kumar, 2006)
and CSF (Noel et al., 2006) when compared to control group.
Within the morphine sub-groups, the rapid progressors
sustained the lowest diversity and divergence, whereas there
were no significant differences between the normal progres-
sors and the controls. A similar pattern of evolution was also
found within the 5′ end of SIV env encompassing V1 and V2
(Tirado and Kumar, 2006). In the present study, we examined
the effect of morphine in the evolution of the 3′ end of SIV
env within plasma and cerebrospinal fluid (CSF) to establish
a correlation with disease progression. We wanted to
determine whether changes in V3 to V5 regions associated
with disease progression.
Analysis of env evolution within the entire region encom-
passing V3–V5 revealed that the virus in plasma showed
generally the same trend, inverse correlation with disease
progression, as that seen in previous studies with tat and env
V1–V2 region. However, in CSF there was a direct correlation
with disease progression, with the morphine-dependent maca-
ques exhibiting greater diversity and divergence that the control
group. Analysis of the individual regions also revealed
differences in evolution between the two compartments, with
plasma showing significant differences within V3 and V4 and
CSF mainly within V4. A consistent pattern does emerge
between the two compartments in the V4 region. In both plasma
Fig. 3. Diversity and divergence of env variable regions 3 to 5 in plasma and CSF from morphine-dependent and control macaques. The env sequences in plasma (A)
and CSF (B) from morphine-dependent and control macaques were aligned and used to calculate diversity and divergence using MEGA 3.1. The mean percent
diversity and divergence are plotted for each monkey. (A) Overall diversity and divergence of env in plasma. (B) Overall diversity and divergence of env in CSF. Error
bars indicate mean of standard error.
379 V. Rivera-Amill et al. / Virology 358 (2007) 373–383
and CSF, the rapid progressors exhibited higher diversity when
compared to control macaques. We have also found a direct
correlation with disease progression in the frequency of amino
acid mutations within V4. The rapid progressors showed higher
mutation frequency as compared to control macaques and this
difference was significant at the earlier time point (12 weeks). A
possible explanation for greater diversity within V4 in rapid
progressors is that this group always maintained a very high
viral load in both plasma and CSF as compared to normal
progressors and control macaques (Kumar et al., 2006). In
addition, two of the three rapid progressors showed progressive
neurological signs including jerky movements, increased
aggressiveness, ataxia, and drooling. These results suggest
that changes within V4 may be related to disease progression,
perhaps through modification of co-receptor use as has been
suggested by others (Cho et al., 1998; Hu et al., 2000; Smyth
et al., 1998).
This study provides evidence that there are remarkable
differences between morphine-dependent and control macaques
indicating a contribution of morphine in both the pathogenesis
Fig. 4. Diversity and divergence of env variable 4 in plasma and CSF over time from morphine-dependent and control macaques. The env V4 sequences in plasma and
CSF from morphine-dependent and control macaques at 12 and 18 wpi were aligned and used to calculate diversity and divergence using MEGA 3.1. The mean
percent diversity and divergence are plotted for each monkey. (A) Over time diversity; (B) Over time divergence. Error bars indicate mean of standard error.
380 V. Rivera-Amill et al. / Virology 358 (2007) 373–383
of SIV/SHIV infection and env evolution. Further analysis of
envelope and other genes in various compartments may provide
a better understanding of the accelerated form of the disease in
Establishment of morphine addiction has been previously
described (Kumar et al., 2004a, 2006; Noel and Kumar, 2006).
Briefly, morphine dependence was established by injecting
increasing doses of morphine (1 to 5 mg/kg of body weight over
a 2-week period) by the intramuscular route at 8-h intervals. The
animals were maintained at three daily doses of morphine
(5 mg/kg) for an additional 18 weeks. All macaques were
infected by the intravenous route with a 2-ml inoculum
containing 10450% tissue culture infective doses each of
simian–human immunodeficiency virus SHIVKU-1B(Singh et
al., 2002), SHIV89.6P(Reimann et al., 1996a; Reimann et al.,
1996b), and SIV/17E-Fr (Flaherty et al., 1997). The animals
were monitored for a period of 20 weeks. Blood was collected
into EDTA vacutainer tubes. The cerebrospinal fluid was
collected by inserting a 23-gauge needle at the junction of the
spinal cord and brain. The clear liquid was spun down at
3000 rpm for 10 min. The supernatant was collected and used
for RNA extraction. The experimental protocol was approved
by the Institutional Animal Care and Use Committee, and the
research was conducted in accordance with the Guide for the
Care and Use of Laboratory Animals.
Amplification, cloning and sequencing of V3–V5 regions of env
Viral RNAwas isolated from plasma and CSF samples at 12
and 18 weeks using the QIAmp UltraSens Viral RNA Mini Kit
(Qiagen, Inc., Valencia, CA) according to the manufacture's
recommendations. A 905-bp fragment of the env region of SIV/
17E-Fr was amplified in a reverse transcription polymerase
chain reaction (RT-PCR) using the One-Step RT-PCR kit
(Qiagen, Inc., Valencia, CA). The oligonucleotide primers
used in the RT-PCR were 5′-TTGTGCACCTCCAGGTTATG-
3′ (nucleotides 7299–7318) and 5′-TTTGTGCTAGGGTTCT-
TGGG-3′ (nucleotides 8204–8185). The RT-PCR reaction
included initial reverse transcription at 50 °C for 30 min and
denaturation at 95 °C for 15 min followed by 40 cycles of
as well as a final extension at 72 °C for 10 min. The 905-bp
fragment was confirmed by agarose electrophoresis prior to
Carlsbad, CA). The insert was confirmed by performing a PCR
using the DNA from transformed Escherichia coli and the
same primer set as described above. Clones were sequenced
using the M13 forward site on pCR2.1 using Big-Dye
terminator chemistry on the Applied Byosystems 3100
Genetic Analyzer, by the DNA Sequencing Facility of Florida
State University, Department of Biological Sciences.
Sequence analysis and statistics
All sequence files were first manually verified and edited as
necessary using the software ChromasLite 2.0 (Technelysium
Mean frequency of SIVenvelope mutations in plasma and CSF by variable region from morphine-dependent and control macaques
Mean frequency of mutations by variable regiona
No. of clonesV3V4 V5 No. of clonesV3 V4V5
aMean frequency of mutation by variable region was calculated as the total number of amino acid mutations per variable region in a monkey at a given time point
divided by the number of clones.
381V. Rivera-Amill et al. / Virology 358 (2007) 373–383
Pty Ltd., Australia). Edited sequences were aligned using
BioEdit version 18.104.22.168 (Hall TA) and Clustal W (Thompson et
al., 1994). The Clustal W program (runs within BioEdit) was set
to perform multiple sequence alignments using the default
penalties. Aligned sequences were used to calculate pairwise
DNA distances and to generate phylogenetic trees using the
program MEGA version 3.1 (Kumar et al., 2004b). All
phylogenetic analyses used the SIV/17E-Fr inoculum sequence
as a reference. Statistical comparisons were done using a one-
tailed t-test. The statistical cut-off for significance in these
analyses was p=0.05. Sequences in this report are available
from GenBank with accession numbers from DQ784853 to
We thank Dr. Martin Hill, Department of Physiology and
Pharmacology, Ponce School of Medicine for insightful
discussions. We acknowledge the support of the Molecular
Biology Core and the RCMI Publications Office (Grant #
2G12RR003050-21). This work was supported by National
Institute on Drug Abuse (DA015013) and National Institute on
Alcohol and Alcoholism (AA015045).
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