The interdomain linker region of HIV-1 capsid protein is a critical determinant of
proper core assembly and stability
Jiyang Jianga, Sherimay D. Ablanb, Suchitra Derebaila,1, Kamil Hercíka, Ferri Soheilianc, James A. Thomasd,
Shixing Tange, Indira Hewlette, Kunio Nagashimac, Robert J. Gorelickd, Eric O. Freedb, Judith G. Levina,⁎
aSection on Viral Gene Regulation, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health, National Institutes of Health, Building 6B,
Room 216, 6 Center Drive, Bethesda, MD 20892-2780, USA
bVirus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute Frederick, Frederick, MD 21702-1201, USA
cImage Analysis Laboratory, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA
dAIDS and Cancer Virus Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA
eLaboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA
a b s t r a c t a r t i c l e i n f o
Received 26 July 2011
Returned to author for revision
17 August 2011
Accepted 14 September 2011
Available online 26 October 2011
HIV-1 capsid protein
In vitro assembly
The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker
(residues 146–150), the function of which remains to be defined. To investigate the role of this region in virus
replication, we made alanine or leucine substitutions in each linker residue and two flanking residues.
Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A,
and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no
viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype. Infectivity of P147L
and S149A is rescued specifically by pseudotyping with vesicular stomatitis virus envelope glycoprotein.
Moreover, despite having unstable cores, these mutants assemble WT-like structures and synthesize viral
for proper assembly and stability of cores and efficient replication.
Published by Elsevier Inc.
The capsid protein (CA) of human immunodeficiency virus type 1
(HIV-1) has a major role in virus assembly and early postentry events
and is derived from the multidomain Gag polyprotein precursor
(Pr55gag) (Freed, 1998; Vogt, 1997). During or shortly after budding of
immature particles, Gag is cleaved by the viral protease to generate the
individual mature viral proteins including (from the N- to C-terminus)
matrix, CA, nucleocapsid (NC), and p6, as well as two spacer peptides
(SP1 and SP2) (Henderson et al., 1992; reviewed in Adamson and
Freed, 2007; Ganser-Pornillos et al., 2008; Swanstrom and Wills, 1997).
Once Gag is cleaved, dramatic structural rearrangements of the proteins
occur (virus “maturation”), resulting in the formation of mature, infec-
tious virions containing electron-dense conical cores (de Marco et al.,
2010; Gross et al., 2000; von Schwedler et al., 1998; Wiegers et al.,
1998; reviewed in Adamson and Freed, 2007; Briggs and Kräusslich,
2011; Ganser-Pornillos et al., 2008).
The HIV-1 core is a fullerene cone composed of approximately 250
hexamers and 12 pentamers that cap both ends of the cone (Ganser
et al., 1999; Li et al., 2000; Pornillos et al., 2011). The N-terminal do-
main (NTD) of CA forms the hexameric lattice and each hexamer is
linked to six others by interactions with C-terminal CA dimers (Briggs
et al., 2006; Ganser et al., 1999; Ganser-Pornillos et al., 2004, 2007;
Huseby et al., 2005; Lanman and Prevelige, 2005; Lanman et al.,
2003; Li et al., 2000; Pornillos et al., 2009). The interior of the core
is a ribonucleoprotein complex containing NC, reverse transcriptase
(RT), integrase (IN), Vpr, Nef, two copies of genomic RNA, and
tRNALys3(Thomas and Gorelick, 2008; Vogt, 1997).
Following entry of the virus into the cell, the core disassembles,
i.e., the CA shell is removed and the ribonucleoprotein complex is
released, resulting in the formation of reverse transcription complexes
is still not clear (reviewed in Arhel, 2010; Levin et al., 2010) and has
Virology 421 (2011) 253–265
⁎ Corresponding author. Fax: +1 301 496 0243.
E-mail address: firstname.lastname@example.org (J.G. Levin).
1Present address: Department of Biochemistry, National University of Singapore,
#02-03 Singapore 117597, Singapore.
0042-6822/$ – see front matter. Published by Elsevier Inc.
Contents lists available at SciVerse ScienceDirect
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been reported to be: (i) soon after viral entry (Fassati and Goff, 2001;
Grewe et al., 1990; Hulme et al., 2011); (ii) several hours postentry
(McDonald et al., 2002; Sayah et al., 2004; Shi and Aiken, 2006;
Stremlau et al., 2006); or(iii)justpriortonuclearimportofpreintegra-
tion complexes (Arhel et al., 2007). Both proper assembly and disas-
sembly of viral cores as well as optimal core stability are essential for
reverse transcription and virus replication (Bowzard et al., 2001; Fitzon
et al., 2000; Forshey et al., 2002; Reicin et al., 1996; Tang et al., 2001,
2003; von Schwedler et al., 1998, 2003).
(Hatziioannou et al., 2004; Keckesova et al., 2004; Stremlau et al., 2004;
Yap et al., 2004) or TRIMCyp, a fusion protein in which a region in
TRIM5α has been replaced by cyclophilin A (CypA) (Brennan et al.,
CA. This results in premature uncoating of the viral core, degradation of
CA, abrogation of reverse transcription, and loss of infectivity (Black
and Aiken, 2010; Rold and Aiken, 2008; Sebastian and Luban, 2005;
by which TRIM5 functions relies on pattern recognition (Ganser--
Pornillos et al., 2011; Pertel et al., 2011) and the ability of TRIM5 to
promote innate immune signaling (Pertel et al., 2011).
pendently folded domains, an NTD (residues 1–145) and a C-terminal
domain (CTD) (residues 151–231), which are connected by a short,
flexible linker (146–150) (Berthet-Colominas et al., 1999; Gamble et
al., 1997; Gitti et al., 1996; Momany et al., 1996) that becomes more
structured upon CA multimerization (Lanman et al., 2003). It has been
both domains can have similar interactions in the CA hexamer and
pentamer (Pornillos et al., 2011). Despite advances in our knowledge
of CA structure and the considerable sequence conservation of the
linker residues, only limited information on their biological and molec-
ular properties has been available thus far. In the case of murine leuke-
mia virus (MLV), several linker mutations inserted into the CA domain
of Gag (i.e., extending the length of the interdomain linker) led to a
defect in virusassemblyandreleaseaswellastheappearanceofvirions
with aberrant morphology (Arvidson et al., 2003).
Recently, the phenotypes of mutations in two residues in the HIV-1
region of interest have been described. One of these residues is Y145,
which in a number of structural studies has been assigned to helix 7
in the NTD (Gamble et al., 1996; Gitti et al., 1996; Momany et al.,
1996; Pornillos et al., 2009). Surprisingly, a new high resolution NMR
structure of HIV-1 CA consisting of residues 144–231 has shown that
Y145 is crucial for the formation of the CTD dimer interface (Byeon et
al., 2009) (also see below). To assess the biological significance of
this finding, two Y145 mutants (replacement with A or F) were con-
structed and were foundto exhibit a severelycompromisedphenotype,
including loss of infectivity (Byeon et al., 2009). Other work on CA
residues S108, S149, and S178, which have the potential to be phos-
phorylated, showed that an S149A mutant has low infectivity (Brun
et al., 2008; Wacharaporninetal.,2007) and is only partially defective
in other assays (Brun et al., 2008).
Inthe presentwork, we set out toinvestigate thebiological function
of all of the residues between the NTD and CTD, i.e., S146–I150, as well
as the flanking residues Y145 and L151 (Fig. 1). Alanine substitution
mutations were made for each of the residues with the exception of
P147, which was changed to leucine. Two mutants (S146A and
T148A) behave like WT, whereas three other mutants (Y145A, I150A,
and L151A) are noninfectious and exhibit severe defects in core assem-
bly and stability and viral DNA synthesis in infected cells. Two mutants
(P147L and S149A), while poorly infectious, have a novel, attenuated
phenotype. Thus, despite the instability of P147L and S149A cores,
these mutants retain an ability to undergo reverse transcription (albeit
less efficiently than WT) and can assemble conical cores that resemble
those of WT virions. These results clearly demonstrate that the interdo-
main linker region has a critical role in facilitatingpropercore assembly
and stability, which in turn ultimately impact the ability of the virus to
undergo efficient replication.
Single amino acid substitutions in the interdomain linker region of HIV-1
The aim of this study was to examine the function of residues
146–150 in HIV-1 replication and in particular, to determine whether
Fig. 1. HIV-1 CA hexamer and monomer illustrating the positions of residues in the interdomain linker region. Residues in this region are shown in red. In both the monomer and
hexamer structures, the NTD and CTD are highlighted in purple and green, respectively. The positions of the five linker residues and the two flanking residues that were mutated to
Ala or Leu are shown in the monomer diagram. Structures were derived from PDB ID: 3GV2 from Pornillos et al. (2009), using Cn3D 4.3 macromolecular structure viewer (NCBI).
J. Jiang et al. / Virology 421 (2011) 253–265
they are important for proper virus assembly. At the outset, we
also decided to include residues Y145 and L151, which flank the inter-
domain linker region. Single alanine substitutions were made in all
residues except P147, which was changed to leucine (Fig. 1), since a
P147A mutant was unable to produce virus particles (i.e., RT activity
in the supernatant fluid of transfected cells was not detectable). The
mutations were introduced into the WT HIV-1 pNL4-3 clone (Adachi
et al., 1986) or the env−derivative pNL4-3KFS (Freed and Martin,
1995; Freed et al., 1992). A diagram of the CA structure highlighting
mer is shown in Fig. 1.
Virus production and infectivity of mutants
To determine whether the CA mutants are able to produce virus
particles, the supernatant fluids of transfected cells were assayed for
RT activity (Freed et al., 1995). As shown in Table 1, column 2, all of
the mutants produced significant amounts of particles and even the
lowest producer, L151A, exhibited 50% of WT activity. When infectivity
of env+virus was measured, it was observed that mutants S146A and
T148A had significant levels of infectivity, similar to those of the WT,
whereas P147L and S149A had only 4–5% of WT infectivity (column
3). In contrast, infectivity was at background level for Y145A, I150A,
There are instances in which retroviral infectivity can be rescued
or enhanced if env−particles are pseudotyped with a heterologous
envelope protein, such as the vesicular stomatitis virus envelope gly-
coprotein (VSV-G) (Aiken, 1997; Brun et al., 2008; Emi et al., 1991;
Jorgenson et al., 2009; Khan et al., 2003; Naldini et al., 1996; Yee et
al., 1994). Although it is known that VSV-G pseudotyped particles
enter via pH-dependent endocytosis (Matlin et al., 1982) rather than
by direct fusion at the plasma membrane, the underlying mechanism
forrescue isstillnotunderstood. Nevertheless,this assaycangiveinfor-
mation on subtle differences between mutants.
mutants, i.e., Y145A, I150A, and L151A, could not be rescued by VSV-G
pseudotyping (Table 1, column 4). The env+mutants that were infec-
tious, i.e., S146A and T148A, had WT levels of infectivity when env−
virions were pseudotyped with VSV-G. Surprisingly, despite the poor
infectivity of P147L and S149A bearing HIV-1 Env, when pseudotyped
(86% and 102%, respectively).
can also occur in a different target cell type, the experiment was
repeated using the TZM-bl assay for infectivity (Table 2) (Derdeyn
et al., 2000; Platt et al., 1998; Wei et al., 2002). This assay utilizes a
HeLa-derived indicator cell line that supports VSV-G-mediated infec-
observation); a positive read-out is therefore unlikely to be simply the
result of “saturating” the infectivity assay. In this case, we found that
ity was rescued almost completely (~80%) (Table 2). This suggests that
at least with respect to this parameter, P147L may be somewhat more
defective than S149A. Importantly, rescue was highly specific for pseu-
dotyping with VSV-G (Table 2). For example, MLV Env was unable to
rescue the infectivity of P147L and S149A, despite the high level of in-
fectivity of “WT” HIV-1 env−virions pseudotyped with MLV Env in
TZM-bl cells (data not shown). Collectively, the VSV-G infectivity data
suggest that the phenotypes exhibited by P147L and S149A differ
from those of the three noninfectious mutants.
Viral protein composition in cell and viral lysates
It was also of interest to determine the viral protein composition
in cell and viral lysates. Cell lysates were prepared as described in
Materials and methods and were subjected to Western blot analysis
with HIV-1 neutralizing serum, which reacted strongly with Pr55gag,
Gag cleavage product p41, IN, and CA (Fig. 2A). To examine the pro-
tein composition of WT and mutant virions, viral lysates were ana-
lyzed with antisera specific for RT, IN, CA, and CypA. Strong bands
representing p66/p51 (RT), IN, CA, and CypA (Fig. 2B) were visual-
ized after probing by Western blot, whereas Pr55gagbands, detected
with anti-CA antibodies, were very weak (Fig. 2C). This indicates
that the mutants do not have major processing defects.
To determine the relative amount of CypA in virions, the band in-
tensities of CypA to IN were quantified; the ratio of CypA to IN was
determined and multiplied by 100 (Fig. 2D). IN was chosen for nor-
malization, since the virion-associated IN is unlikely to be affected
by the mutations in CA (Tang et al., 2003). The WT value was set at
100%. All of the mutants except for Y145A had approximately WT
levels of CypA. However, the Y145A mutant had ~5-fold less CypA
than WT, suggesting that this mutant is even more defective than
the other noninfectious mutants.
Transmission electron microscopy (TEM)
of HIV-1 particles. To investigate the architecture of mutant cores, WT or
mutant virions were imaged by TEM (Fig. 3). In addition to conical cores
(arrows), centric and acentric cores (cores that are proximal to the viral
membrane) were also observed. Infectious HIV-1 is expected to have a
ing the core structures in a large number of particles (ranging from ~100
to ~400), we determined that for WT and infectious mutants S146A and
T148A, the percentage of conical cores was between 40 and 45%
Virus production and infectivity.
VirusVirus production (%)a
aVirus production was assayed as RT activity in the supernatant of transfected cells.
Values were converted to percentage of WT level for each of three or more indepen-
dent transfections and were averaged.
bInfectivity was measured in the single-round LuSIV assay (Roos et al., 2000). WT
infectivity was set at 100%.
cHIV-1 env−virions were pseudotyped with VSV-G envelope protein, as described
in Materials and methods.
dNumbers to the right of the “±” represent the standard deviation.
Rescue of infectivity of pseudotyped WT and mutant env−virions is Env-specific.a
Virus HIV-1 (pIIINL4env) A-MLVenv
et al., 2000; Platt et al., 1998; Wei et al., 2002). WT infectivity was set at 100%. Three
independent experiments were performed. The data shown are from a representative
experiment, in which each sample was assayed in triplicate.
bNumbers to the right of the “±” represent the standard deviation.
J. Jiang et al. / Virology 421 (2011) 253–265
(Table 3). Conical cores were detected in P147L and S149A virus popula-
tions at a frequency approximately half that of the WT. However, as is
from thoseofauthentic WT cores.The noninfectious mutants lacked con-
acentric cores, as also observed in earlier studies (Auerbach et al., 2006;
Dorfman et al., 1994; Fitzon et al., 2000; Forshey et al., 2002; Reicin et
al., 1996; Scholz et al., 2005; Tang et al., 2001, 2007; von Schwedler et
al., 1998, 2003).
Another approach to evaluate the assembly competency of Gag
mutants is to perform in vitro assembly reactions with purified viral
proteins (Campbell and Rein, 1999; Campbell and Vogt, 1995; Ehrlich
et al., 1992; von Schwedler et al., 1998). Here, we purified the CA pro-
teins of WT, P147L, and S149A, as well as the Y145A and I150A CA
proteins, which were chosen to represent the class of noninfectious
mutants that form only aberrant cores (Fig. 3). The in vitro assemblies
were analyzed by TEM (Fig. 4). As is typical for WT CA, long tubular
structures were formed (Ehrlich et al., 1992). The P147L and S149A
assemblies had structures that were similar to WT. In some reactions,
subtle differences were observed between the WT and these mutants
(e.g., formation of somewhat shorter tubes by P147L and elongated
narrow tubes by S149A). What is particularly striking, however, is
that in contrast to these CA proteins, neither Y145A nor I150A CA
formed any recognizable structures.
Taken together, the EM data demonstrate that while the noninfec-
tious mutants are severely defective with respect to virus assembly
both in vivo and in vitro, mutants P147L and S149A exhibit an atten-
uated assembly phenotype.
Synthesis of viral DNA in infected cells and in detergent-treated virions
Several studies have shown that a defect in conical core assembly
is associated with an inability to synthesize viral DNA in infected cells
(Brun et al., 2008; Fitzon et al., 2000; Reicin et al., 1996; Rulli et al.,
2006; Tang et al., 2001). It was therefore of interest to determine
the efficiencies of viral DNA synthesis in infections with WT or each
of the P147L, S149A, and I150A mutants. Quantitative PCR (qPCR)
was used to detect the major products of reverse transcription:
R-U5, (−) strong-stop DNA; U3–U5, minus-strand transfer DNA;
Gag, late minus-strand DNA; R-5′UTR, plus-strand transfer DNA;
and full-junction (FJ), 2-long terminal repeat (2-LTR) circles, which
are a marker for entry of viral DNA into the nucleus (Fig. 5A).
The data showed that P147L and S149A synthesized ~10-fold less
of each viral DNA product compared to WT. The results for the two
mutants were essentially the same, although it appears that S149A
made more 2-LTR circles than P147L. With I150A, the amount of
DNA products synthesized was ~104lower than WT and the two
late products were not detectable. This suggests that the inability to
synthesize viral DNA in I150A-infected cells is likely due to the com-
plete absence of normal cores in the virion population (Fig. 3;
Table 3). Collectively, the results demonstrate a dramatic contrast be-
tween the levels of viral DNA detected in cells infected with P147L
and S149A and the noninfectious I150A.
We also examined DNA synthesis in detergent-treated virions by
using the endogenous RT (ERT) assay. The amounts of early (R-U5)
and late minus-strand (Gag) products were measured using qPCR
(Fig. 5B). Samples were taken before the initiation of the ERT assay
% of WT
Incorporation of CypA
Fig. 2. Analysis of WT and mutant viral proteins present in cell lysates and in virions. HeLa cells were transfected with WT or mutant plasmid DNAs. Proteins present in cell and
virion lysates were separated by SDS-PAGE in 10% gels and were detected by Western blot analysis. (A) Cell-associated viral proteins probed with HIV-1 neutralizing serum.
Note that analysis of cell-associated proteins suggests that I150A might have a processing defect, but importantly, this defect was not detected in an anti-CA blot of the viral lysate
(see (C), below). (B) Virion-associated proteins probed sequentially with a mixture of anti-CypA and anti-IN, anti-RT, and anti-CA antibodies. (C) Entire Western blot analyzed with
anti-CA antibodies. The CA bands are the same as those shown in (B). (D) Relative incorporation levels of CypA into WT and mutant virions. The CypA and IN band intensities were
quantified as described in Materials and methods. The data were normalized for each sample by calculating the ratio of the level of CypA to the level of IN and then multiplying by
100. The WT ratio was set at 100%. Note that in this figure and in Figs. 5, 7, and 8, error bars indicate the standard deviation from at least two or three independent experiments.
J. Jiang et al. / Virology 421 (2011) 253–265
(pre-ERT) and 24 h later. In this case, following incubation for 24 h,
WT and all of the mutant samples showed the same levels of viral
DNA synthesis for both early and late products of reverse transcrip-
tion. This finding is consistent with previous studies demonstrating
that the absence of conical cores does not block reverse transcription
in the ERT assay (Brun et al., 2008; Kaplan et al., 1994; Tang et al.,
Assay of CA function in vivo
To learn more about the ability of the CA mutants to function in
the infected cell, we performed an abrogation assay to test whether
the CA mutants could saturate the post-entry defect in owl monkey
kidney (OMK) cells conferred by TRIMCyp, a potent host restriction
Fig. 3. TEM analysis of WT and mutant virions present in extracellular fluids of transfected HeLa cells. Env−virions were used for this experiment. Mutants are identified above each
image. The arrows point to representative virus particles in the field with conical cores. The scale bars are 100 nm.
Analysis of HIV-1 WT and mutant virions by TEM.
Virus No. of
No. of particles whose cores are:
aNumbers to the right of “±” represent the standard deviation.
bNumbers in parentheses are percentages of the total number of particles.
J. Jiang et al. / Virology 421 (2011) 253–265
factor that interacts with HIV-1 CA and blocks virus infection. In our
experiments, OMK cells were infected with WT and mutants P147L,
S149A, and I150A. After brief (4 to 6 h) incubation, the infected cells
were challenged with an HIV-1-green fluorescent protein (GFP) re-
porter virus. If the CA protein of the initial virus is functional, then
TRIMCyp will be saturated and the reporter virus should be able to
replicate. As a consequence, a significant number of the infected
cells should register as GFP+(Fig. 6). In fact, this is what we observed
when WT was used as the initial virus. If mutant CA proteins are not
able to interact with TRIMCyp, we should obtain the opposite result.
In this case, almost no infectivity was observed when the mutants
were tested. In some experiments, there was a very small difference
between the activity of P147L and S149A compared with the activity
of I150A, but in other experiments, no difference was detected.
These results show that the three mutants were all essentially in-
active in the TRIMCyp abrogation assay. Moreover, the data further
indicate that the differences between the two poorly infectious mu-
tants (i.e., P147L and S149A) and the noninfectious mutants that
were observed in other assays (see above) do not affect the assay
for CA-TRIMCyp interaction.
Determination of viral core stability
In view of the fact that TRIM5 proteins interact most efficiently
with CA present in an intact core, i.e., assembled CA (Black and
Aiken, 2010; Ganser-Pornillos et al., 2011; Kar et al., 2008; Langelier
et al., 2008; Sebastian and Luban, 2005; Stremlau et al., 2006; Zhao
et al., 2011), it seemed possible that the results of the TRIMCyp
assay mightreflect common defects in thecore stability of the mutants.
Optimal core stability is an important determinant of core function;
virions with hyperstable or unstable cores are not infectious (Bowzard
et al., 2001; Forshey et al., 2005, 2002; Tang et al., 2003; von Schwedler
et al., 2003). To address this issue, we determined the amounts of CA
and RT associated with core structures. WT and mutant virions were
treated with 0.2% (vol/vol) Triton X-100 prior to sedimentation in su-
crose gradients. If detergent concentrations greater than 0.2% (even as
low as 0.3%) were used, only low amounts of CA protein could be
detected for all of the mutants with the exception of S146A and
T148A, which have a WT phenotype (Table 1). This suggested that in
the case of the other five mutants, viral cores are detergent-sensitive
For analysis of CA content, detergent-treated particles were sedi-
mented in sucrose step gradients; cores were found in fractions 3, 4,
and 5 (Tang et al., 2003). Fractions were probed by Western blot
(Fig. 7A) and the ratio of the amounts of CA in fractions 3 to 5 to
total CA was calculated and multiplied by 100 (Fig. 7B). WT, S146A,
and T148A had ~40% of total CA associated with the core, whereas
Y145A, I150A, and L151A had barely detectable amounts. For P147L
and S149A, between 3 and 5% of total CA was recovered in the core
RT content of virions was measured by sedimenting detergent-
treated virions in 20 to 70% (wt/wt) linear sucrose gradients followed
by assay of RT polymerase activity (Freed et al., 1995) (Fig. 8A). Viral
cores, which sediment in sucrose gradients with a density of 1.24–
1.28 g/ml, were found in fractions 8 to 10. WT, S146A, and T148A
each had between 15 and 20% of total RT in core fractions, whereas
the noninfectious mutants had ≤1% RT in cores (Fig. 8B). Mutants
P147L and S149A showed broad peaks of activity, in contrast to the
sharp peak seen for WT (Fig. 8A). The percentage of RT associated
with the cores of these mutants was between 5 and 10% of total RT ac-
tivity (Fig. 8B).
These results show that mutants P147L and S149A retain minimal
amounts of CA and somewhat higher levels of RT in core fractions.
This suggests that the cores of these mutants are also unstable, al-
though not to the same extent as the cores of the noninfectious mu-
tants. Thus, there appear to be small, but reproducible differences
between the core stability of P147L and S149A compared with the
core stability of the noninfectious mutants. Apparently, these small
differences do not affect the outcome of the TRIMCyp assay, al-
though as seen above, other properties of P147L and S149A are
clearly distinguishable from the corresponding properties of the
noninfectious mutants in a variety of assays including infectivity
In the present study, we sought to investigate the biological role of
residues between the two domains of HIV-1 CA (Y145-L151) (Fig. 1)
Fig. 4. Analysis of in vitro assemblies of WT and mutant CA proteins by TEM. Purified CA proteins were assembled and then processed for TEM, as described in Materials and
methods. Mutants are identified above each image (Y145A, I150A) or pair of images (WT, P147L, S149A). The scale bars are 100 nm.
J. Jiang et al. / Virology 421 (2011) 253–265
and in particular, to assess their importance for virus assembly and
replication. Our approach was to make point mutations in residues
encompassing the entire region, i.e., alanine substitutions in all resi-
dues except P147, which was changed to leucine. The results clearly
demonstrate that the CA interdomain linker is crucial for facilitating
proper core stability and architecture.
Analysis of mutant phenotypes shows that the mutants can be
divided into three classes: (i) infectious virions (S146A, T148A);
(ii) completely noninfectious virions with severe defects in viral mor-
phology and function (Y145A, I150A, L151A); and (iii) virions with
very low infectivity, having an attenuated phenotype (P147L, S149A)
(Table 1). Interestingly, the infectivity data for some of the mutants
can be correlated with what is known about CA structure in this region.
Recently, a study combining high resolution NMR analysis of recombi-
nant HIV-1 CA containing residues 144–231 with cryo-EM data for
tubular assemblies of this protein revealed the importance of residues
idue Y145 is crucial for the formation of this interface and for biological
function, since Y145A or Y145F mutants are not infectious and have
defects in core stability and assembly (Byeon et al., 2009), consistent
with our Y145A data (Table 1; Figs. 2–4, 7 and 8). Moreover, of all the
mutants studied here, Y145A is the only one that exhibits a CypA de-
ficiency(Fig. 2D), presumably as a result of globalchanges in CA fold-
ing. A similar defect was also observed with two severely defective
N-terminal CA mutants (W23A and F40A) (Tang et al., 2003). A sig-
nificant reduction in CypA incorporation would be expected to be as-
sociated with mutated residues in the NTD, although our data do not
rule out such an effect when mutation of a residue near the NTD also
resultsinglobal disruptionofCA structure. Theapparentdiscrepancy
concerning the localization of Y145 is likely due to differences in the
techniques andprotein constructs
Residues L151 and T148 (in order of importance) also exhibit
intermolecular interactions that are involved in CTD dimer formation
(Byeon et al., 2009; I.L. Byeon, personal communication). Thus, the
lack of infectivity of L151A is consistent with the structural data
(Table 1). Most likely, retention of the WT phenotype in the case of
T148A (Table 1) is due to the conservative change from threonine
to alanine. The S146A mutation may not impact infectivity because
this residue is exposed on the surface of the protein (A.M. Gronenborn,
As mentioned above, a major conclusion that emerges from our
study is that residues Y145–L151, with the possible exception of
S146, are critical for proper core formation. Indeed, point mutations
that lead to abrogation or severe reduction of infectivity are often as-
sociated with formation of virion cores that are highly unstable: re-
tention of CA in core fractions is negligible (Y145A, I150A, L151A)
or only slightly higher than background (P147L, S149A), whereas
~40% of total CA in the sample is associated with WT, S146A, and
T148A cores under our conditions (Fig. 7). Core instability of S149A
was also observed in studies on HIV-1 CA residues that are potential
substrates for phosphorylation (Brun et al., 2008; Wacharapornin
et al., 2007). Reduction of RT activity associated with unstable mutant
cores is less severe than depletion of CA in the case of P147L and
S149A (Fig. 8). Thus, removal of the mutant CA shells might occur
shortly after entry by premature uncoating (Stremlau et al., 2006;
reviewed in Arhel, 2010; Levin et al., 2010), whereas RT might be
less labile due to its location in the interior of the virus. In accord
with the CA sedimentation data, we also find that P147L, S149A,
and I150A are unable to saturate TRIMCyp in the assay for abrogation
used for thestructure
DNA copies per cell
DNA copies per virion
WT P147LS149A I150A
R-U5 Gag R-U5Gag R-U5Gag R-U5Gag
Fig. 5. Analysis of DNA synthesized in WT- and mutant-infected cells and in ERT assays
with detergent-treated particles. The mutants used for the assays were P147L, S149A,
and I150A. Viral DNA from infected cells and from detergent-treated virions was quan-
tified as described in Materials and methods. (A) Analysis of viral DNA in infected cells.
The data are plotted in bar graphs as DNA copies per cell for the following viral DNA
products synthesized by RT: R-U5 (−) strong-stop DNA, closed; U3–U5, minus-strand
transfer DNA, gray; Gag, late minus-strand DNA, slanted stripe; R-5′UTR, plus-strand
transfer DNA, open; FJ 2-LTR circles, horizontal stripe. The amounts of DNA were nor-
malized for the number of cells recovered and for the number of viruses used to infect
the cells. (B) Viral DNA synthesis in ERT assays. The data are expressed as DNA copies
per virion. Values for the R-U5 and Gag DNA products are plotted in bar graphs.
Amount of DNA products present in virions (i.e., intravirion DNA) prior to the ERT
assay, closed; amount of DNA products after the ERT assay, open.
0 100200 300400
% GFP+ cells
Fig. 6. TRIMCyp abrogation assay. An ELISA assay was used for measuring CA concentra-
tions in the virus preparations (Tangand Hewlett, 2010). The ability of WT and three mu-
tants (P147L, S149A, and I150A) to interact with TRIMCyp expressed in OMK cells was
ted against CA concentration (ng/well). Three independent were performed with similar
results. The data shown are from a representative experiment.
J. Jiang et al. / Virology 421 (2011) 253–265
of host restriction (Fig. 6), which gives a positive readout only if the
cores have optimal stability (Forshey et al., 2005).
A striking result of this work is the novel finding that despite the
poor replication capacity of P147L and S149A, infectivity can be res-
cued in an efficient and specific manner by pseudotyping env−virions
with VSV-G (Tables 1 and 2). Similar results were obtained for S149A
and S178A in one of the studies of CA phosphorylation (Brun et al.,
2008). Additionally, these authors showed that the pseudotyped
virions undergo more efficient viral DNA synthesis. In contrast, VSV-G
pseudotyping does not rescue the infectivity of the three noninfectious
mutants in our study (Table 1).
It is generally thought that HIV-1 entry into cells depends on in-
teraction with cellular receptors followed by fusion at the plasma
membrane (Hunter, 1997). However, there is also evidence support-
ing the possibility that entry occurs by an endosomal pathway
(Miyauchi et al., 2009). With respect to pseudotyping with VSV-G, it
is known that VSV-G-mediated virus entry occurs via pH-dependent
endocytosis (Matlin et al., 1982), but the reason why this mode of
entry can result in rescue of infectivity is not known. We speculate
that endocytosis might allow delivery of the cores to the nuclear
pore without premature uncoating and a requirement to traverse cy-
toplasmic microfilament/microtubule networks (Arhel et al., 2007;
Bukrinskaya et al., 1998; McDonald et al., 2002), thereby bypassing
the core instability defect. A somewhat similar suggestion was made
in an earlier study on VSV-G pseudotyping of nef-defective HIV-1
virions (Aiken, 1997). Alternatively, some other aspect of VSV-G-
mediated entry, e.g., faster fusion kinetics with VSV-G than with
HIV-1 Env (Hulme et al., 2011; Iordanskiy et al., 2006), might result in
bypass of the infectivity defect conferred by the P147L and P149A mu-
tations. Collectively, these considerations lead us to suggest that the
two mutants might be useful tools for studies on the viral entry
The P147L and S149A assembly properties and core architecture
are also of great interest. Thus, despite the instability of P147L and
S149A cores (Fig. 7), a significant number of mutant virions contain
conical cores that are indistinguishable from WT structures in images
generated by TEM (Fig. 3; Table 3). Moreover, in contrast to the
Y145A (Fig. 4) (Byeon et al., 2009), Y145F (Byeon et al., 2009), and
I150A (Fig. 4) CA proteins, P147L and S149A CA form tubular assem-
blies that are very similar to the long tubes assembled by WT CA,
although some subtle differences can be detected by TEM (Fig. 4). Ex-
in an effort to identify potential ultrastructural differences between WT
and mutant core structures.
It is now established that in addition to other defects, virions having
unstable or hyperstable cores are unable to undergo or complete
reverse transcription in infected cells (Aiken, 2006; Bowzard et al.,
2001; Forshey et al., 2002; Tang et al., 2001, 2003; von Schwedler et
al., 2003). Accordingly, we find that the noninfectious mutant I150A
makes ~104less viral DNA products than WT and no late products
(Fig. 5A). However, in agreement with the EM analysis, synthesis of
viral DNA products by the P147L and S149A mutants is reduced by
only 10-fold compared with WT and all of the expected DNA products
are detected (Fig. 5A).
Taken together, our results raise the following question: “How can
we reconcile our finding that in two assays affected by core stability,
P147L and S149A give negative results (Figs. 6 and 7), whereas in
other assays reflecting core structure, assembly, and retention of RT
in cores (Figs. 3, 4, 5, and 8), these mutants exhibit an attenuated
phenotype?” It would appear that differences between the WT and
mutant CA protein structures, which in turn dictate core structure
and biological activity (reviewed in Adamson and Freed, 2007; Vogt,
1997), are subtle and may not be sufficient to generate a positive
readout in some assays. This is most likely the case in the TRIMCyp ab-
rogation assay (Fig. 6). Data from a recent structural study support the
idea that the hexagonal scaffold of TRIM5α proteins is assembled
using the symmetry and spacing of the CA lattice as a template
(“pattern recognition”) (Ganser-Pornillos et al., 2011). Thus, even
stability (Forshey et al., 2005) could prevent significant binding of
TRIM5αproteins toCA. Additionally, the lowrecovery of CA from iso-
lated mutant cores is not entirely unexpected,since sedimentation at
high speeds, even for relatively short times, is likely to lead to disrup-
tion of cores that are unstable. In contrast, EM and PCR analyses of
DNA synthesisinvolve moregentletreatment ofvirions, thereby gener-
ating a modulated response in these assays.
These findings have important implications for understanding the
molecular nature of HIV-1 assembly, since they underscore the unusual
plasticityof CA,whichdespitetherigorous structuralrequirementsthat
govern assembly and integrity of viral cores, permits some expression
of biological activity even under less than optimal circumstances
(Tang et al., 2007). Additional studies on the structure of the P147L
and S149A CA proteins should be invaluable for elucidating the ultra-
structure of HIV-1 conical cores and its relation to CA function.
1 2 3 4 5
% of Total CA
Fig. 7. Retention of CA in core fractions of WT and mutant virions. Virions treated with
0.2% Triton X-100 (vol/vol) were subjected to sedimentation in sucrose step gradients.
Aliquots of each fraction were subjected to Western blot analysis by using anti-CA
serum. (A) HIV-1 CA bands are shown for WT and mutant samples. (B) The band inten-
sities of CA in each fraction were determined as described in Materials and methods.
The values for fractions 3, 4, and 5 (detergent-resistant core fractions) were combined
and are shown in bar graphs as the percentage of total CA protein recovered from the
J. Jiang et al. / Virology 421 (2011) 253–265
the NTD and CTD of HIV-1 CA (Y145-L151) have a crucial role in virus
assembly and formation of conical cores. Mutations in three of these
residues (Y145A, I150A, and L151A) lead to a total loss of infectivity,
defects in core morphology and stability, as well as virtually com-
plete abrogation of viral DNA synthesis. However, two mutants
(P147L and S149A), while poorly infectious, exhibit an attenuated
phenotype, including rescue of infectivity by pseudotyping with
VSV-G, modest ability to undergo reverse transcription, and assem-
bly of cores with seemingly normal architecture. These findings pro-
vide new insights into the biological function of a region in HIV-1 CA
that has onlyrecentlybecome the subject of intense interest and rep-
resents a potential target for anti-HIV therapy.
Materials and methods
Cell culture, transfection, and RT assay
OMK cells (a generous gift from Christopher Aiken, Vanderbilt
University Medical Center, Nashville, TN), HeLa cells, and 293 T cells
with 10% fetal bovine serum (Hyclone), 2 mM glutamine, penicillin
(50 IU/ml),andstreptomycin(50 μg/ml).Threecelllineswereobtained
from the AIDS Research and Reagent Program, Division of AIDS, NIAID,
NIH: (i) LuSIV cells (from Jason W. Roos and Janice E. Clements, catalog
no. 5460) (Roos et al., 2000), which were grown in RPMI 1640 medium
with 300 μg/ml of hygromycin B (Invitrogen, Carlsbad, CA) and the
same supplements as described above; TZM-bl cells (from John C.
Kappes, Xiaoyun Wu, and Tranzyme Inc., catalog no. 8129) (Derdeyn
et al., 2000; Platt et al., 1998; Wei et al., 2002), which were grown in
the same medium used for HeLa cells (see above); and (iii) HeLa
CD4+cells (HeLa CD4 Clone 1022 from Bruce Chesebro, catalog no.
1109), which were maintained as recommended (Chesebro and
Wehrly, 1988; Chesebro et al., 1990, 1991).
To produce HIV-1 virions, HeLa cells were transfected in 100-mm
dishes with10 μg ofWTor mutant proviralDNA,using Lipofectamine™
2000 (Invitrogen), according to the manufacturer's instructions. Super-
natant fluids werecollected36 to 48 h after transfection.Cellulardebris
was removed by low-speed centrifugation and subsequent passage of
the clarified fluids through 0.45-μm-pore-size syringe filters. The fluids
RT Activity (x10 )
RT Activity (x10 )
RT Activity (x10 )
% of Total RT
1 2 3 4 5 6 7 8 9 101112
1 2 3 4 5 6 7 8 9 101112
1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112
1 2 3 4 5 6 7 8 9 101112
1 2 3 4 5 6 7 8 9 101112
1 2 3 4 5 6 7 8 9 101112
1 2 3 4 5 6 7 8 9 101112
Fig. 8. Retention of HIV-1 RT in WT and mutant viral cores. Virions were treated with 0.2% Triton X-100 (vol/vol) and were sedimented through 20% to 70% (wt/wt) linear sucrose
density gradients (Tang et al., 2003). Aliquots of each fraction were assayed for RT activity. (A) RT activity of WT and mutant fractions plotted in a bar graph. (B) RT activity in core
fractions (fractions 8, 9, and 10, underlined) was quantified. The data are plotted as the percentage of total RT activity in the gradient. The value for percent RT activity in L151A core
fractions was at the detection limit and was therefore subtracted from the values for all of the other samples.
J. Jiang et al. / Virology 421 (2011) 253–265
were adjusted to pH 7.3 with HEPES buffer (final concentration,
10 mM) and were divided into several tubes prior to storage at
−80 °C. Particle production was determined by an exogenous RT
assay (Freed et al., 1995). For ERT assays, virus was prepared by trans-
fecting 293 T cells with mutant or wild-type pNL4-3-based proviral
plasmids using the calcium phosphate method (Julias et al., 2001). In
et al. (1990).
Single-cycle infectivity assays
Viral infectivitywas measured usinga single-cycle assay. Equivalent
amounts of virus particles, as determined by RT assay, were used to in-
fect LuSIV cells (Roos et al., 2000). Luciferase activity was measured
using the Luciferase Assay System kit from Promega (Madison, WI),
according to the manufacturer's instructions. Some single-cycle assays
were performed by using TZM-bl indicator cells (Derdeyn et al., 2000;
Platt et al., 1998; Wei et al., 2002). The Env vectors used for pseudotyp-
ing HIV-1 env−virions were as follows. The VSV-G expression vector
pHCMV-Genv (Yee et al., 1994) was a generous gift from Jane Burns
(University of California at San Diego School of Medicine, La Jolla, CA).
The HIV-1 Env expression vector pIIINL4env has been described pre-
viously (Murakami and Freed, 2000). The plasmid pSV-A-MLVenv
(Landau et al., 1991), which encodes amphotropic (A) MLV Env,
was obtained from Dan Littman and Nathaniel Landau through the
AIDS Research and Reference Reagent Program, Division of AIDS,
Plasmids and site-specific mutagenesis
The plasmids used to make site-specific mutations in the CA linker
coding region were as follows: (i) HIV-1 pNL4-3 (GenBank accession
no. AF324493) (Adachi et al., 1986); and (ii) env−subclone, pNL4-
3KFS, which has a frameshift mutation at a KpnI site in the env coding
region (Freed and Martin, 1995; Freed et al., 1992). PCR products
were generated by nested PCR with appropriate primers. The prod-
ucts were digested with BamHI and SphI, so that the BamHI–SphI frag-
ment in pNL4-3 could be replaced by the corresponding fragment
containing the desired mutation. The same restriction sites were
used to transfer mutations into the env−pNL4-3KFS proviral vector.
In each case, the mutation was confirmed by sequencing performed
by AGCT, Inc. Plasmid DNAs were prepared with a QIAFilter Maxi
Kit (QIAGEN Inc.—USA, Valencia, CA). The HIV-1 GFP reporter con-
struct in which nef was replaced by the coding region for GFP was a
generous gift from Christopher Aiken (He et al., 1997; Shi and
Western blot analysis of WT and mutant viral proteins
Cell and viral lysates were prepared and fractionated in 10% SDS-
polyacrylamide gels as described previously (Freed and Martin,
1994; Huang et al., 1995). A chemiluminescence kit (SuperSignal
West Dura Extended Duration Substrate), anti-human horseradish
peroxidase (HRP), and anti-rabbit HRP were purchased from Pierce
(division of Thermo Fisher Scientific, Inc.). HIV-1 neutralizing serum
(catalog no. 1983 and 1984) and anti-CA sera (HIV-1SF2p24 antise-
rum, catalog no. 4250) were obtained from the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH. Rabbit
anti-CypA was purchased from Biomol International (Plymouth
Meeting, PA). Rabbit anti-HIV IN and RT sera are described in an ear-
lier publication (Klutch et al., 1998). Band intensities were quantified
by first scanning the film and then converting the image into a jpg file
that could be analyzed by using NIH Image (http://rsbweb.nih.gov/
Preparation of purified CA proteins expressed in Escherichia coli
expressed in E. coli Rosetta (DE3) pLysS (Novagen) with a C-terminal
His tag, using a CA construct as well as an expression and purification
protocol (with some modifications), both kindly provided by Michael
Summers, University of Maryland Baltimore County, Baltimore, MD
(Tang et al., 2002). The final eluate from the cobalt affinity column
(TALON Superflow Metal Affinity Resin, Clonetech, Palo Alto, CA) was
dialyzed against 50 mM sodium phosphate buffer, pH 7.2, and 5 mM
Transmission electron microscopy
HeLa cells transfected with WT and mutant virions were fixed
with 2% glutaraldehyde in 0.1 M sodium cacodylate and examined
by TEM as described (Freed and Martin, 1994; Tang et al., 2001). For
in vitro assembly, purified CA protein (60 to 100 μg) was incubated
in 1 M NaCl at 4 °C overnight. Reactions were analyzed by TEM as de-
scribed above for virus-infected cells.
Assay of viral DNA in infected cells and in detergent-treated virions (ERT)
Virus produced by transfecting 293 T cells was used to infect HeLa
et al., 2001). Primers and probes used for quantitation of viral and cellu-
lar nucleic acids have been described (Buckman et al., 2003; Thomas
et al., 2006). Briefly, viral sequence targets of the primers and probes
used for quantitation of viral DNA included (−) SSDNA (R-U5), minus-
strand transfer product (U3–U5), late minus-strand synthesis product
(gag), plus-strand transfer product (R-5′UTR), FJ (i.e., 2-LTR circles),
gag for genomic RNA, and CCR5 to determine cell equivalents present
in extracts from infected HeLa CD4+cells. Quantities of viral DNA were
normalized for cell recovery and exogenous template RT activity.
For ERT reactions, virus produced from transfections was sequen-
tially treated with DNase I (Sigma-Aldrich, St. Louis, MO) and then
subtilisin A (Sigma-Aldrich) (Thomas et al., 2008); ERT assays were
performed as described (Thomas et al., 2008). Viral DNA and RNA
species were quantified using the primers and probes mentioned
above. The copies of viral DNA were normalized to the number of vi-
rions present in the corresponding pre-ERT sample (calculated by as-
suming 2 copies of genomic RNA per virion).
CA (p24) ELISA assay
Supernatant fluids from cells transfected by WT and mutant virus-
es were assayed for p24 by a Europium nanoparticle immunoassay
(ENIA), as described previously (Tang and Hewlett, 2010). Briefly,
each well of the 96-well microtiter plate was coated with 60 μl of
monoclonal anti-HIV-1 p24 antibody (final concentration, 2 μg/ml)
(catalog no. C65690M, Meridian Life Science, Inc., Saco, ME) and
was incubated overnight at 4 °C. After washing five times with buffer
containing PBS and 0.05% Tween 20 (PBST) (each new step was always
preceded by washing with PBST), the plate was blocked by adding
350 μl of blocking buffer (Starting Block™ Blocking Buffer, Thermo
Fisher Scientific, Inc.) to each well. One hundred microliter each of
p24 standards (ranging from 0.05 ng/ml to 1 ng/ml) and experimental
sampleswereaddedandwerethenincubatedfor1 hat37 °C.Notethat
RT buffer containing 0.05% Nonidet P-40 (Freed et al., 1995) and subse-
quently making serial dilutions in PBST containing 1% bovine serum
albumin (BSA). Addition of 100 μl per well of the detection antibody,
Waltham, MA), was followed by incubation for 1 h at 37 °C. Finally, 107
europium nanoparticles conjugated with streptavidin in PBST contain-
ing 1% BSA were added and the plates were incubated for
J. Jiang et al. / Virology 421 (2011) 253–265
30 min at 37 °C. Fluorescence was measured with a Victor fluorometer
(PerkinElmer) as described (Tang and Hewlett, 2010). The concentra-
tion of HIV-1 p24 in the supernatants was determined based on the
standard curve obtained in the experiments.
Abrogation of host restriction assay
Assays to determine the ability of WT and mutant viral CA to re-
strict infection in OMK cells expressing TRIMCyp, a fusion of TRIM5α
and CypA (Nisole et al., 2004; Sayah et al., 2004), were performed es-
sentially as reported previously (Forshey et al., 2005; Shi and Aiken,
2006). All virus samples were assayed for CA (p24) content so that
lutions of WT or mutant virus pseudotyped with VSV-G were used to
infect OMK cells and were incubated for 4 to 6 h at 37 °C. HIV-1-GFP
reporter virus pseudotyped with VSV-G was then added to the
infected cells and incubated for 24 h at 37 °C. The reporter virus was
subjected to preliminary titration on OMK cells and an amount giving
less than 1% of total cells registered as GFP+was chosen for the assay.
In the final step, thecells were treated withtrypsinand werefixedin a
freshly prepared solution of 4% paraformaldehyde in PBS. GFP expres-
sion was analyzed with a Becton–Dickinson FACScan flow cytometer.
Isolation of viral cores
For analysis of CA and RT retention in viral cores, supernatant
fluids collected from transfected HeLa cells were sedimented at
100,000×g. The pellets were resuspended in PBS and Triton X-100
(final concentration 0.2% (vol/vol)) was added. Samples were then
subjected to sucrose gradient centrifugation without prior incubation.
For CA, step gradients were used, since the CA yield for all samples
was higher and more reproducible after centrifugation for 2 h, com-
pared with the amounts detected following overnight centrifugation
(required for linear gradients). Five fractions were collected from
the top of the tube and the amount of CA in each fraction was mea-
sured by Western blot analysis using anti-CA serum. Fractions 1 and
2 represent the soluble and detergent-soluble fractions, respectively;
fractions 3, 4, and 5 represent detergent-resistant core fractions (Tang
et al., 2003).
Toanalyze RTretention in viral cores, detergent-treated virions (see
above) were sedimented through 20% to 70% (wt/wt) linear sucrose
gradients as described (Tang et al., 2003). (Assay of enzymatic activity
is more sensitive than Western blotting and in this case, sufficient
amounts of activity were detected after overnight centrifugation.)
Twelve 0.4-ml fractions were collected from the top of the gradient
and were assayedforexogenous RTactivity. The density of thefractions
was determined as described in an earlier report (Tang et al., 2003).
Cores were found in fractions with a density of 1.24 to 1.28 g/ml
(Fig. 8A, fractions 8–10).
We thank Christopher Aiken for his generous gift of OMK cells and
the HIV-1-GFP reporter construct aswell asforhelpful adviceregarding
the TRIMCyp assay, Michael Summers for kindly providing a CA con-
struct and protocol for bacterial expression and purification of HIV-1
CA, Jane Burns for the VSV-G expression vector (pHCMV-Genv), and
the AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH for antisera, cells, and plasmids, as detailed in the text. We
are also indebted to Angela M. Gronenborn and In-Ja L. Byeon for valu-
able discussion. This work was supported in part by the Intramural Re-
search Program at the National Institutes of Health (Eunice Kennedy
Shriver National Institute of Child Health and Human Development
[J.J., S.D., K.H., and J.G.L.] and the National Cancer Institute, Center for
Cancer Research [S.D.A. and E.O.F.]) and in part by the Office of Science
and Health Coordination, Food and Drug Administration, National
Heart, Lung, and Blood Institute, National Institutes of Health (S.T. and
I.H.).Thisprojecthasalso beenfunded inwhole or in part with federal
funds from the National Cancer Institute, National Institutes of
Health, under contract HHSN261200800001E with SAIC-Frederick,
Inc. (F.S., K.N., J.A.T., and R.J.G.). The content of this publication
does not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by the
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