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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1906-1915
Human Immunodeficiency Virus Type 1 Vpr Alters Bone Marrow Cell
Function
By
Joseph Kulkosky,
Alexey Laptev,
Shubhra Shetty,
Alagarsamy Srinivasan,
Mohamad BouHamdan,
Darwin J. Prockop, and
Roger J. Pomerantz
From the Dorrance H. Hamilton Laboratories, Center for Human
Virology, Division of Infectious Diseases, Department of Medicine,
Jefferson Medical College, and the Department of Microbiology and
Immunology, Thomas Jefferson University, Philadelphia, PA; and the
Center for Gene Therapy, Allegheny University of the Health Sciences,
Hahnemann Division, Philadelphia, PA.
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ABSTRACT |
Vpr, a 96 amino acid protein, encoded by the human immunodeficiency
virus type I (HIV-1), is important for efficient infection of
mononuclear phagocytic cells. These cells are abundant in whole bone
marrow, which can easily be cultured in vitro to support hematopoiesis.
Our experiments indicate that Vpr plays a role in the potent activation
of murine and human mononuclear phagocytic cells within a hematopoietic
microenvironment. In murine cultures, avid erythrophagocytosis is
triggered by transduction of marrow cells with supernatant derived from
PA317 cells transfected with a murine retroviral delivery vector
bearing a Vpr expression cassette. Supernatants derived from cells
transfected with the same vector carrying sequences for the expression
of other relevant viral and nonviral proteins do not induce
erythrophagocytosis to any marked degree. The effect on human marrow
cells is similar, where treatment promotes adhesion of mononuclear
phagocytic cells to culture plates in association with other nucleated
and nonnucleated cells that undergo subsequent engulfment. The
differential effects of Vpr point and deletion mutants in both marrow
culture systems fortify the view that the effect is specific to HIV-1
Vpr. Addition of low molar quantities of purified Vpr to marrow
cultures is also capable of promoting cell adhesion and phagocytosis,
suggesting that extracellular Vpr is the effector of the phenomenon.
Accelerated phagocytosis is a hallmark of promonocyte, monocyte, and
macrophage activation and its occurrence within a hematopoietic
microenvironment may account for critical in vivo pathogenic features
of HIV-1 infection. First, activation of mononuclear phagocytes may
promote productive viral infection; and second, premature phagocytosis could provide, at least in part, a molecular explanation for the induction of the idiopathic cytopenias that are typical of individuals infected with HIV-1.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN IMMUNODEFICIENCY virus type I
(HIV-1) replication occurs primarily in cells of hematopoietic
origin,1,2 and the most severe pathogenic features
associated with infection can ultimately be attributed to either
malfunction or destruction of subsets of cells derived from this
lineage, eg, CD4+ T lymphocytes and monocyte/macrophages.
Accordingly, the use of whole bone marrow cultures was pursued as an in
vitro model system that might be manipulated easily to obtain a more
clear understanding, particularly of early events, that underlie
hematologic disorders associated with long-term retroviral
infection.3 Specifically with regard to patients with
acquired immunodeficiency syndrome (AIDS), these disorders typically
include anemia, lymphocytopenia, monocytopenia, and neutropenia. Almost
all patients with advanced AIDS exhibit pancytopenia, with greater than
90% suffering from anemia.4 Most current studies indicate
that hematopoietic stem cells are refractory to HIV-1 infection and
must undergo at least modest differentiation to express critical
surface receptors to permit virus entry and subsequent
replication.5 Several reports have focused on HIV-1
infection of auxillary cells in bone marrow that are required to
support normal hematopoiesis; however, the target cell populations and
the ability of virus to replicate in cells susceptible to infection in
the bone marrow compartment are conflictory.6-8 In
addition, the influence on hematopoiesis and the maturation of stem
cells within the marrow compartment in the presence of active viral
replication is also relatively obscure. Some studies have demonstrated
deficiencies or imbalance in the release of growth factors from
HIV-1-infected marrow-derived cells required to support normal
hematopoiesis.9 This could account for the onset of the
variety of cytopenias that occur in HIV-1-infected individuals. As has
previously been reported,10 it also seemed reasonable,
however, to begin with the premise that perhaps expression or release
of a specific HIV-1 gene product, or perhaps a limited combination of
viral proteins, could perturb or alter marrow stem cell function. The
effects of direct transduction of marrow cultures with specific
expression vectors for various proteins, including the HIV-1 regulatory
and accessory proteins such as Tat, Rev, and Vpr, was the focus of this
study. The effects of transduction for Vpr expression was of particular
interest as this protein has been implicated in the efficient infection and replication of HIV-1 in mononuclear phagocytes. In these
experiments, transduction of whole bone marrow propagated in vitro with
a Vpr expression vector initiated what appears to be premature or
enhanced phagocytosis of cells in the marrow cultures by mononuclear
phagocytes. The effect was also observed after addition to marrow
cultures of recombinant Vpr either as a fusion to
glutathione-S-transferase (GST) or as a free protein released from the
GST fusion partner by protease treatment. Accelerated phagocytosis
could reasonably account for certain of the cytopenias typical in vivo
in persons suffering from advanced stages of AIDS.
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MATERIALS AND METHODS |
Plasmid construction.
An HIV-1NL4-3 Vpr open reading frame DNA fragment was
cloned into an amphotropic murine retroviral delivery vector, SLX-CMV, using compatible 5' BamHI sites and a filled in 3'
HindIII site of the insert into a 3' Hpa I site
of the vector to create the Vpr expression plasmid, SLX-CMV-Vpr.
Subsequent sequencing verified the integrity of the construct.
HIV-189.6 Vpr point and deletion mutants were obtained by
polymerase chain reaction (PCR) amplification from existing plasmids
(provided by A.S.), using Vent polymerase and primers bearing 5'
BamHI and 3' Mlu I sites for direct insertion into the vector, SLX-CMV. All plasmids were sequenced through the Vpr
insert before use. Construction and use of other vectors such as
pLX-Tat, pLX-Rev,11 and SLX-CMV-CAT12 have been
described previously.
Bone marrow culture transduction.
PA317 cells, a murine retroviral packaging line, were transfected with
10 to 15 µg of plasmid DNA per 100 × 20 mm dish at 20%
confluency. After media change within 12 to 16 hours, supernatant was
harvested 48 hours post-transfection, passed through 0.45-µm filters,
and stored at  80°C until application to marrow cultures. Murine and human marrow cultures were initiated by seeding aspirates at
a density of 2 × 106 nucleated cells per 35 × 10 mm dish. Murine cells were maintained in Dulbecco's modified
Eagle's medium (D-MEM) with 10% fetal bovine serum
(FBS), whereas human cells were grown in D-MEM with 20% FBS. Media was
typically replaced 1 to 3 days after plating and 1 mL/dish of
supernatant from transfected PA317 cells was applied within 1 week of
seeding plates. Adherence and subsequent phagocytosis of resident cells
or added latex beads by mononuclear phagocytes occurred typically
within 24 to 48 hours after supernatant addition. Cultures were then
either photographed directly or washed once with phosphate-buffered
saline (PBS), fixed carefully with 100% methanol, and photographed
through an inverted light microscope.
Reverse transcriptase assay.
Sixty microliters of PA317 cell supernatant was adjusted to 1% Triton
X-100 and added to a final reaction volume of 150 µL containing 40 mmol/L Tris-HCl, pH 7.3, 100 mmol/L KCl, 5 mmol/L MgCl2, 1 µg poly (rA)-oligo (dT), 10 mmol/L dithiothreitol (DTT), and 2.5 µmol/L 3H dTTP. Reactions were incubated for 1.5 hours, spotted onto DE81 filters, dried, and counted with scintillation.
GST-Vpr fusion protein expression, purification, and treatment of
marrow cultures.
The construction of the pGEX-GST-Vpr expression plasmid has been
described previously.13 Bacterial expression
of the fusion protein was initiated by induction of at least 1 L of
cells with 0.1 mmol/L isopropyl  -D-thiogalactopyranoside (IPTG) for
1 hour. The induced bacterial cells were harvested by low speed
centrifugation and the cells were suspended in 10 mL of PBST (20 mmol/L
phosphate buffer, pH 7.3, 150 mmol/L NaCl, 1% Triton-X-100) containing
2 mmol/L EDTA, 0.4 mmol/L phenylmethylsulfonyl flouride, 2 µg/mL leupeptin, 50 µg/mL of 1-chloro-3-tosylamido-7-amino-2-heptone, and
100 µg/mL of L-1-tosylamido-2-phenylethyl-chloromethyl ketone. The
cells were disrupted by sonication and the lysate cleared by
centrifugation at 10,000g for 10 minutes at 4°C. The
supernatant was applied to a 1 mL column of glutathione agarose
pre-equilibrated with TBST (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L
NaCl, and 0.5% Tween 20). Vpr was released from the GST after the
addition of thrombin (Sigma, St Louis, MO) to GST-Vpr at 4 U/mg of fusion protein and incubation for 2 hours at room temperature.
The digested sample was applied to 1 mL of glutathione agarose and free
Vpr was collected in the column flow-through fraction. The Vpr
containing fraction was dialyzed against 4 × 500 mL of PBS at
4°C and sterilized by filtration through a 0.2-µm low
protein-binding filter. GST-Vpr or Vpr in PBS was added directly to
marrow cultures at a approximate final concentration of 3 nanomolar,
which is consistent with the in vivo levels as reported previously by
others.14
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RESULTS |
From previous studies, retroviral delivery vectors were available that
contained various expression cassettes, including some specific for
certain HIV-1 proteins such as viral transcriptional transactivator,
Tat, and HIV-1 Rev. As shown in Fig 1, a
murine leukemia virus transduction vector, SLX-CMV-Vpr, was
subsequently constructed that expresses the 96-amino acid HIV-1
accessory protein, Vpr, independent of other viral products. This
protein seemed to be an important candidate for study of its effect on
bone marrow cultures, because it has been reported to be capable of
cell-cycle arrest15 and differentiation16 and
can be found free of virions in the serum of HIV-1-infected
individuals.14
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| Fig 1.
Schematic of the HIV-1 Vpr transduction vector.
pSLX-CMV-Vpr encodes a Vpr expression cassette from HIV-1 (strain
NL4-3) downstream of the cytomegalovirus (CMV) promoter
(pCMV). The retroviral packaging cell-line, PA317, provides, in trans,
viral proteins necessary for vector transfer.30 Below, the
linear map of Vpr, 96 amino acids, denotes the approximate location of
domains important for Vpr function.31,32
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Erythrophagocytosis induced in murine marrow cultures.
Because mouse bone marrow aspirates are obtained more readily than
human samples, the effects of Vpr as well other proteins were initially
assessed after transduction of murine whole marrow cultures in vitro.
First, the retroviral delivery vectors were introduced into PA317
cells, a murine packaging cell line, by calcium-phosphate-mediated
transfection. Forty-eight hours afterwards, supernatants containing
Moloney murine leukemia virus (MoMuLV) virions were harvested and
applied to murine marrow cells. Supernatants were introduced after the
initiation of hematopoietic colony formation, which, under our plating
conditions, occurs within a few days to 1 week of seeding the
cultures.17 Within 36 to 48 hours after application of
supernatant to the murine cultures, an easily detectable gross
morphological change was observed. As shown in
Fig 2, media derived from cells transfected
with a Vpr expression cassette elicited avid erythrophagocytosis that
is seen to a significantly lesser extent in cultures transduced with
vector alone, SLX-CMV, or vector containing other expressed proteins
such as the chloramphenicol acetyl-transferase (CAT) gene (not shown)
or even HIV-1 Tat and Rev (not shown). The effect is not observed when
supernatants from transfected NIH3T3 cells are added to marrow
cultures. NIH3T3 is the parental line for PA317 cells and therefore
does not possess the MoMuLV packaging function. This indicates that
virion release plays some role in facilitating Vpr action in the
system.
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| Fig 2.
Effect of transfected PA317 cell supernatants on murine
marrow cultures propagated in vitro. (A) Media from PA317 cells
transfected with vector alone or transfected with (B) vector with HIV-1
Vpr expression cassette or (C) vector with HIV-1 Tat expression
cassette. E, free erythrocyte; M, erythrocyte-coated mononuclear
phagocyte. The results shown are typical of at least three independent
experiments.
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As shown in Fig 2B, erythrophagocytosis is easily visible as adherent
cells of the promonocyte/monocyte/macrophage lineage become coated
entirely by much smaller, light bright erythrocytes. After attachment,
red blood cells (RBCs) are then engulfed completely within the next 12 to 24 hours. Because the attachment and phagocytosis of erythrocytes
was so obvious and extensive in these cultures (1) the nature of this
effect, (2) whether it was mediated by expression and perhaps release
of Vpr either from the packaging cell-line or directly from
transduced marrow cells, and (3) finally its biological
relevance to HIV-1 infection became obvious issues to be addressed further.
Vpr-associated erythrophagocytosis is similar to that induced by
lipopolysaccharide (LPS).
Phagocytosis is known to be a hallmark of promonocyte, monocyte, and
macrophage activation.18,19 To assess the nature of the
Vpr-associated effect and whether it might be similar to the state of
activation triggered by other inducers, marrow cultures were treated
with LPS, referred to typically as endotoxin. As shown in
Fig 3, treatment of cultures with LPS
recapitulated the gross morphological features observed in our
supernatant addition experiments. As shown in Fig 3, increasing amounts
of LPS added directly to the culture media up to 30 ng/mL lead to
greater numbers of mononuclear phagocytes cells displaying active
endocytosis. This observation supported our initial hypothesis that the
Vpr-associated effect indeed reflected a natural pathway of phagocytic
cell activation.
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| Fig 3.
Effect of LPS addition on murine marrow cultures.
Cultures initiated as described in previous figure were treated with
LPS (Sigma; catalogue no. L-2143) at the concentrations as indicated.
After 24 hours, cultures were washed once with PBS and photographed. E,
free erythrocyte; M, erythrocyte-coated mononuclear phagocyte; B,
blanket cell or stromal cell with large thin cytoplasm and
endothelial-like in morphology.
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The differential effect of Vpr mutants on the induction of
phagocytosis.
Next, it seemed important to demonstrate convincingly that Vpr was the
critical factor required to initiate, either directly or indirectly,
activation of adherent, phagocytic cells within the marrow cultures. To
that end, a library of Vpr point mutants and HIV-1 Vpr HXB2, a
frameshift mutant at residue 72 of the protein, were transferred into
the SLX-CMV transduction vector (see Fig 1). As was performed in our
initial experiments, the constructs were introduced into PA317 cells
and the media derived from the transfections was applied to duplicate
cultures of murine marrow cells. After 48 hours, the cultures were
washed, fixed, and assessed for the extent of erythrophagocytosis. As
shown in Table 1, indeed our collection of
mutants displayed a range in their ability to activate phagocytes. H71C
induced at least wild-type (WT) levels of activity. R62S, L64S, and
C76A appeared intermediate, whereas the HXB2 frameshift and A59P
mutants were typically lower in their ability to induce
erythrophagocytosis. As shown in Table 1, the supernatants were also
assayed for reverse transcriptase (RT) activity to determine whether
the sheer number of virions varied drastically among the sample
collections. This is a concern, because virions themselves appear
capable of inducing activation to some extent. However, RT activity
only varied a few fold among the supernatants, thus alleviating the
concern that MoMuLV particles were a sole determinant in activating
phagocytes. In addition, the effects after direct application of
sucrose gradient purified HIV-1 NL4-3 WT and
NL4-3  Vpr virions, derived from transfection of 293 cells, was assessed. Both were capable of eliciting
erythrophagocytosis; however, WT virus showed marked acceleration of
the process relative to Vpr deletion mutant virions (data not shown).
Thus, collectively, the differential behavior of a series
of Vpr mutants fortified our view that this retroviral protein was
playing a critical role in triggering both the extent and rate of this
process.
The induction of Vpr-associated cell adherence and phagocytosis in
human marrow cultures.
The next series of experiments focused on attempting to demonstrate
mononuclear phagocyte cell activation within human bone marrow
cultures. The approach was multi-faceted. First, human marrow cells
from HIV-1-seronegative individuals were treated with the PA317
supernatants, as was performed for the murine cultures. However, in
this case, nonadherent cells were first washed from the plates and
activation of phagocytes was assessed by addition of 1-µm deep blue
latex beads directly to the culture media. As shown in
Fig 4A, cultures treated with Vpr mutants
A59P and HXB2 showed low levels of uptake of the colored beads, whereas
WT Vpr and H71C supernatants, which triggered erythrophagocytosis in the murine cultures, enhanced markedly the uptake of the latex spheres.
Recapitulation of the erythrophagocytic effect was next tested in human
marrow cultures seeded densely. As shown in Fig 4B, supernatants
derived from WT Vpr and H71C elicited enhanced binding of nonadherent
cells relative to other controls, such as no addition or Vpr A59P and
HXB2 (data not shown), which were demonstrated previously to be
negative or low for erythrophagocytosis when applied to murine
cultures. The human cultures behaved somewhat differently from the
murine treated cells in that engulfment of the attached cells was not
visually apparent, at least in the same time frame.
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| Fig 4.
Effect of transfected PA317 cell supernatants
on human marrow cultures propagated in vitro. Approximately 2 × 106 nucleated marrow cells were plated in D-MEM with
heat-inactivated 20% FBS. Supernatants (1 mL) from PA317 cells
transfected with SLX-CMV bearing expression cassettes for Vpr mutants,
as indicated in the figure, were applied. (A) The following day, cells
were washed with media and 200 µL of latex beads (Sigma; catalogue
no. L-1398) were added directly to cultures replenished with media, and
cultures were incubated at 37°C for an additional 30 minutes,
washed with PBS, and photographed. Dark rounded cells are phagocytes
ingesting deep-blue latex spheres. (B) More densely seeded cultures
retaining large numbers of nonadherent cells were treated with PA317
supernatants as described above, except that no further additions were
made. After 24 hours, cells were fixed with 100% methanol and
photographed. The effects shown are typical of at least three
independent experiments.
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The effect of HIV-1 infection, with or without Vpr, on human marrow
cultures.
An alternative approach was then taken by infecting human marrow
cultures with the HIV-1 clone (NL4-3) that harbors a
functional Vpr sequence or with the same clone containing a deletion in
Vpr (NL4-3 Vpr). Two effects were noted. Within 24 hours
after addition of the virus, many more cells infected with the HIV-1
wild-type Vpr clone became adherent relative to the Vpr deletion
mutant. This effect appeared to be transient. Nevertheless, within 1 week, both cultures showed obvious signs of infection and were strongly positive for virus production by HIV-1 p24 antigen as assessed by
enzyme-linked immunosorbent assay (ELISA-Dupont, Wilmington, DE;
data not shown). Visual inspection, after fixation of the cells 10 days
later, showed obvious gross morphologic differences between the two
cultures. An increase in adherent cells was noted for
HIV-1NL4-3 infection relative to the HIV-1NL4-3
 vpr mutant and those cells, as shown in
Fig 5, displayed increased numbers of
attached light dense erythrocytes and perhaps granulocytes. Macrophage-tropic virus (Bal 9 strain) with or without Vpr was also
tested. Similar but much more modest differences relative to that
observed for infection by the T-lymphocyte-tropic
HIV-1NL4-3 was observed (data not shown). This appeared to
be attributable to a depletion of mononuclear phagocytic cells through
syncytia-formation induced by infection with the macrophage-tropic
strain in these marrow cultures.
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| Fig 5.
Effect of HIV-1 infection on human marrow cultures. Human
marrow cultures were infected with HIV-1 strains NL4-3 or
NL4-3 Vpr (Vpr deletion mutant). The microscope filter
was adjusted to project nonadherent cells as light dense. E, free
erythrocyte or granulocyte; M, mononuclear phagocyte coated with
nonadherent cells.
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The experiments described above may be problematic in the sense that,
without careful study, it is unknown how a wild-type Vpr versus a Vpr
mutant virus may alter the dynamics of hematopoiesis (in particular,
the production of monocyte/macrophages and erythrocytes). Although such did not appear to be the case, the presence of more mononuclear phagocytes and RBCs in one culture versus the other could
lead to a similar observation. Although some caution may be warranted,
these data are nonetheless entirely consistent with previous results
and are important because they relate most directly to events that may
occur during natural HIV-1 infection in vivo.
Direct addition of purified, recombinant Vpr induces cell adherence
and phagocytosis in marrow cultures.
GST-Vpr was expressed in bacterial cells and purified using a
single-step procedure by binding and release from glutathione agarose.13 Vpr was removed subsequently from GST by
treatment with thrombin, whose cleavage site is between the two fusion
partners within the GST-Vpr chimera. Western blotting analysis, shown
in Fig 6, indicates that both GST-Vpr and
free Vpr obtained after protease treatment are the appropriate
molecular weight and intact. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) did not indicate the presence
of other contaminating bacterial proteins (data not shown).
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| Fig 6.
Purification of GST-Vpr and Vpr. The HIV-1 (strain NL4-3)
Vpr coding sequence was cloned into the GST fusion protein bacterial
expression vector, pGEX. The GST-Vpr fusion protein, synthesized after
induction of the bacterial cells with 0.1 mmol/L IPTG, was purified
(lane 1) by binding to glutathione agarose. Free Vpr was obtained by
proteolytic digestion of the GST-Vpr protein and collecting the
flow-through fraction from glutathione resin. The purity was assessed
by Western blotting analysis using a rabbit polyclonal antisera against
Vpr.
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GST-Vpr or free Vpr, added directly to murine marrow cultures at 0.25 µg/mL of media, was sufficient to induce avid erythrophagocytosis by
mononuclear phagocytes, as described previously in the supernatant (Fig
2) or LPS (Fig 3) addition experiments (data not shown). Of note, the
effect of adding the recombinant proteins to human bone marrow cultures
was of greater interest.
As shown in Fig 7A, the addition of
purified GST (0.25 µg/mL culture media) had no effect relative to
untreated marrow cultures. As might be expected due to the presence of
IgG receptors on phagocytic cells, addition to the marrow culture of 1 µL/mL of rabbit polyclonal antisera, raised against Vpr, resulted in
a modest increase in cell adherence to the culture dishes, likely
reflecting some level of activation as shown in Fig 7B. The effect was
much more pronounced when GST-Vpr was added alone or along with the
polyclonal rabbit antisera (Fig 7C and D). There consistently appeared
to be a modest additive effect on marrow cell adherence and subsequent
phagocytosis when the recombinant GST-Vpr protein was added along with
the polyclonal antisera, although this is not evident in the high-power image shown in Fig 7.
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| Fig 7.
Effect of GST-Vpr addition to human marrow cultures.
Purified recombinant proteins were added directly to human marrow
cultures propogated in vitro as indicated in the figure at a final
concentration of approximately 3 nanomolar. Addition was performed in
the absence or presence of rabbit polyclonal antisera directed against
Vpr, as indicated in the figure, using 1 µL of antisera/mL of
media.
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The effect of adding free Vpr to the human marrow cultures was next
evaluated. As shown in the low-power images of
Fig 8, Vpr induced considerable adherence
of erythrocytes and likely other cell types to adherent cells on the
cultures dishes. The cultures were washed with PBS to remove unbound
cells from both the untreated and treated cultures, and cell morphology
was enhanced by reaction with iron stain. As shown in Fig 8B, adherent
cells are typically coated with deeply stained red blood cells. This effect is minimal in the untreated control culture, as shown in Fig 8A.
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| Fig 8.
Effect of recombinant Vpr addition to human marrow
cultures. Purified recombinant Vpr proteolytically cleaved from the
GST-Vpr chimera was added directly to cultures at a final concentration
of approximately 3 nanomolar. Twenty-four hours later, untreated and
treated cultures were washed with PBS and iron stain (Sigma) was
applied to enhance cell morphology.
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DISCUSSION |
Propagation and manipulation of whole bone marrow cultured in vitro has
afforded a novel and tractable system that appears to be well-suited to
study the effects of independently expressed HIV-1 proteins after
transduction as well as the dynamics of HIV-1 infection. The most
important feature of the system specifically with regard to the
activities of Vpr is the preponderance of mononuclear phagocytic cells
in an environment that reasonably recapitulates natural hematopoiesis
and does not require addition of exogenous growth
factors.20 This self-supporting microenvironment
facilitates the production and maturation of mononuclear phagocytes as
well as other cells that may be influenced by the biological properties of Vpr. In this system, Vpr plays a role in the activation of mononuclear phagocytic cells. This was demonstrated in both murine and
human marrow cells and the observation is fortified by the differential
effects of a series of Vpr mutants on these cultures. It is difficult
to correlate the known intracellular activities of Vpr, such as
cell-cycle arrest, with the activation of mononuclear phagocytes that
is observed in the treated marrow cultures. For instance, Vpr HXB2 and
the point-mutants, A59P and H71C, have been reported to be capable of
arresting cells.21 The mutants Vpr HXB2 and A59P do not
trigger erythrophagocytosis efficiently, but Vpr H71C appears to elicit
this effect as well as or perhaps to a greater extent than that of
wild-type Vpr. A larger collection of mutants are being tested
currently to provide additional information as to whether intracellular
functions of Vpr bear any relation to the phenomenon reported herein.
Mononuclear phagocytic cells and CD4+ T lymphocytes
represent the major targets of infection by HIV-1 in vivo. Previous
data demonstrating a specific requirement of Vpr or its homologues for
efficient infection of mononuclear phagocytic cells by HIV-1, HIV-2,
and simian immunodeficiency virus (SIV)22-24 are
interesting in the light of our results. It seems reasonable to
hypothesize that Vpr involvement in the activation of mononuclear
phagocytes might likely enhance productive infection by HIV-1, which is
perhaps analogous to the requirement that quiescent T lymphocytes
undergo exogenous stimulation to initiate active viral
replication.25 This hypothesis is currently being tested.
The precise mechanism of action for HIV-1 Vpr-induced activation is
also being pursued, and some clues are provided by the fact that the
effect is similar morphologically to that triggered by addition of LPS.
It seems reasonable to propose that Vpr is released into supernatant of
transfected PA317 cells during the process of virion budding and
consequently induces activation directly. There is no available
evidence that suggests that Vpr can associate with murine virions,
although this is under investigation currently. It is also possible
that the role of Vpr is secondary, either inducing the expression or
allowing the export of another moiety capable of engaging activation.
We have shown recently that shuttling of a variety of nonviral and
viral proteins into HIV-1 virions is possible when they are fused to a
small 23 amino acid Vpr interactor domain.13 Again, it is
unknown whether this mechanism of egress for Vpr or its potential
interactors can occur from cells during the maturation of murine
particles. It is possible that transduction and expression of Vpr in
certain cells within marrow cultures may occur. The vector SLX-CMV
bears a Neo expression cassette allowing G418 selection after
transduction of marrow cultures. Selection leads to the survival of
large endothelial-like cells referred to as blanket cells (data not
shown). These cells play a critical role in the growth and maturation
of a variety of other cells during hematopoeisis.26 Perhaps
transduction of this specific population of cells results in a
perturbation of the normal cytokine profile in marrow cultures
signaling mononuclear phagocytes to initiate erythrophagocytosis.
Of the possibilities listed above, direct activation by cellular
binding of Vpr appears most likely, although the others outlined above
could occur and contribute to induction of the effect. Nevertheless, the reductionist approach of the protein addition experiments indicates
an identical pattern of activation in both murine and human marrow
cultures after direct addition of soluble, recombinant Vpr.
Interpretation of these experiments must be viewed cautiously due to
possible LPS contamination from disrupted bacterial cells as assessed
by sensitive assays for the presence of endotoxin. However, the data
provided in Fig 3 suggest that rather high levels of LPS (3 to 30 ng/mL) are required to initiate an equivalent level of
erythrophagocytosis as that observed by supernatant addition in the
marrow cultures. Because cell adherence and accelerated phagocytosis
was observed in both murine and human marrow cultures with Vpr
introduced to the marrow cells in three different contexts, it seems
likely that Vpr is the direct effector of mononuclear phagocyte
activation. More interesting is what appears to be accelerated differentiation along the monocyte/macrophage lineage induced by
addition of recombinant Vpr to CD34+ stem cells isolated
from human cord blood (data not shown). Accelerated differentiation
related to Vpr expression has been reported previously for
rhabdomyosarcoma cells.16 Experiments described previously suggest it is likely that Vpr is capable of binding to cells
extracellularly.27 Such a hormone model of Vpr action is
appealing, because release and extracellular uptake of Vpr triggering
activation, and perhaps even differentiation of cells in the process of
maturation, would likely increase the population of cells susceptible
to productive infection of HIV-1. The recent report that extracellular
Vpr can perforate the cytoplasmic membranes of neuronal cells and
induce ion channel flux leading to cell death28 may be
pertinent to our results. Such disturbances in the marrow cultures
could lead to enhanced phagocytosis of damaged cells.
Regardless of the underlying mechanism of activation, our experiments
further provide some molecular explanation, at least in part, for the
induction of cytopenias in HIV-1-infected individuals.29 Although studies have not been directed to ascertain whether
phagocytosis is enhanced in the marrow compartment of
HIV-1-seropositive patients versus uninfected persons, it is quite
obvious that this effect occurs in vitro in both the murine and human
bone marrow cultures. Premature phagocytosis of erythrocytes would lead
to anemia, and the engulfment of nucleated cells during hematopoiesis
could result in a variety of other cytopenias typical of long-term
HIV-1 infection.29
| |
ACKNOWLEDGMENT |
The authors thank Ling-Xun Duan for providing recombinant Vpr and the
plasmids containing the VprNL4-3 expression cassette as
well as pLXN-Tat and pLXN-Rev and thank Didier Trono (Salk Institute,
La Jolla, CA) for kindly providing macrophage-tropic HIV-1Bal
9 infectious clones. Muhammad Amjad kindly provided the protocol
and the reagents for RT assays and Dr Omar Bagasra assisted in the
photography of some of the cultures. We also appreciate Brenda O. Gordon for formatting of the figures, Rita M. Victor for excellent
secretarial assistance, and T.D. Allen (Patterson Institute,
Manchester, UK) for kind review of the manuscript.
| |
FOOTNOTES |
Submitted April 2, 1998; accepted October 28, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Roger J. Pomerantz, MD, Thomas Jefferson
University, 1020 Locust St, Suite 329, Philadelphia, PA 19107; e-mail:
roger.j.pomerantz{at}mail.tju.edu.
| |
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