Download Internalization of Human Immunodeficiency Virus

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RAPID COMMUNICATION
Internalization of Human Immunodeficiency Virus Type I and Other Retroviruses by
Megakaryocytes and Platelets
By D. Zucker-Franklin, S. Seremetis, and Z.Y. Zheng
Direct infection of megakaryocytes and platelets by human
immunodeficiencyvirus type I (HIV-I) or other retroviruses
has not been demonstrated. To determine whether this
could occur, murine bone marrow was co-cultivated with
the amphotropic retrovirus-producing cell line PA317-N2,
and freshly isolated normal human bone marrow and
platelets were co-cultivated with HIV-infected H9 cells. In
each case, ultrastructural analyses showed viruses within
megakaryocytes and platelets. In murine specimens, the
uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent.
These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIVassociateddisease. In addition, these observations suggest
that cells of the megakaryocyte lineage could serve as
target cells in gene transfer experiments using retroviralbased vectors.
0 1990 by The American Society of Hematology.
T
place only at a morphologically unidentifiable precursor
stage is not known. To answer these questions, a simple
approach was used. Mouse bone marrow was co-cultured
with a mouse cell line infected with an amphotropic retrovirus, and freshly isolated normal human bone marrow was
incubated for various time periods with HIV-infected H9
cells. Ultrastructural analyses of the murine specimens
showed that uptake of virus by MK was avid at all stages of
differentiation. On the other hand, interiorization of HIV by
human MK and platelets was comparatively rare, even when
antibody to the virus was present in the incubation medium.
H E QUESTION WHETHER megakaryocytes (MK)
can take up retroviruses is of interest from several
points of view. First, the cell's nucleus undergoes endomitosis, often attaining a ploidy of 62 and even 128 N. Therefore,
if retroviruses were subject to interiorization by MK, integration of exogenous genetic material could be particularly
efficient in this cell lineage. This could be advantageous in
studies designed to effect new gene expression or regulation,
as has been done with other hematopoietic ~e1ls.l.~
Megakaryocytes have not been used previously as target cells in this
system. Yet, defects affecting the function of their progeny,
the platelets, have been genetically defined and may lend
themselves to correction by means of retrovirus-based gene
transfer.
A less salubrious consequence of retrovirus uptake by
mature MK or platelets could be associated with the uptake
of pathogenic viruses, such as the human immunodeficiency
virus type I (HIV-I). It is conceivable that viral interiorization, even at a late stage of MK differentiation, could affect
thrombocytopoiesis. The cause for thrombocytopenia seen in
patients with AIDS is believed to be multifactorial. Fully
differentiated megakaryocytes of such patients have ultrastructural abn~rmalities,~
as well as integrated proviral
sequences6 that probably result in platelet underproduction.
Immune mechanisms appear to be responsible for decreased
platelet survival.' Whether mature megakaryocytes or platelets are able to take up HIV-I or whether such infection takes
From the Department of Medicine, New York University Medical
Center, New York, N Y .
Submitted January 9, 1990; accepted February 26.1990.
Supported in part by US Public Health Service Grants No. A M
12274 and CA 16246.
Z.Y.Z. was supported by a fellowship from the Irvington House
Institute.
Address reprint requests to Dorothea Zucker-Franklin. MD. New
York University Medical Center, 550 First Ave. New York. NY
10016.
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 I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7510-0025$3.00/0
1920
MATERIALS AND METHODS
Preparation of MK. Mouse marrow was flushed from the
femurs of anesthetized 8- to 10-week-oldmale C57 BL/6J mice and
collected into CATCH medium as described in detail before.' After
centrifugation at room temperature for 7 minutes at 400g, the pellet
was washed once, resuspended in medium, and subjected to Percoll
gradient centrifugation to enrich for MK. Human bone marrow
consisted of aliquots of aspirates obtained from individuals who
required marrow sampling for unrelated reasons. If the quantity of
the specimen permitted, it was also subjected to Percoll gradient
centrifugation. Otherwise, the buffy coat of the specimen centrifuged for 7 minutes at 400g was used. Buffy coat or gradient
enriched MK were resuspended in 0.5 mL RPMI 1640 containing
10% fetal calf serum (FCS). In some instances, peripheral blood
platelets prepared from 10 mL platelet-rich plasma were added to
the marrow sample.
Mouse cell cultures containing retrovirus. Details of vector
construction and retroviral packaging have been reported elsewhere!
Briefly, the amphotropic retrovirus used here was produced by
infecting 4 x lo6 PA317 cells'with 5.0 mL of high titer supernatant
from two cloned lines. These lines produce ecotropic retroviruses
bearing pN2," a construct that encodes neomycin resistance. The
PA317-N2 cell lines were propagated in the presence of G418
(GIBCO, Grand Island, NY). Clones producing high-titer amphotropic N2 (>l x lo6colony-forming units [CFU]/mL) were identified
by titering on NIH3T3 (murine fibroblasts) and U20S (human
osteosarcoma, American Type Culture Collection [ATCC]) cells.
Preparation of HIV-infected cells. H9 cell cultures were infected with the I11 R F strain of HIV-I obtained from Dr David Ho."
The infected cells were sedimented at 9,000 rpm at 4°C for 30
minutes. To free HIV of sediment, the supernatant was centrifuged
at 36,000 rpm for 30 minutes, and the cell pellet was resuspended in
RPMI 1640 containing 10% FCS. The sediment-free virus was
added to the H9-HIV-infected cells.
Infection of murine M K . The day before infection, 1 x lo6
Blood, Vol 75, No 10 (May 15). 1990: pp 1920-1923
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UPTAKE OF RETROVIRUS BY MK AND PLATELETS
1921
Fig 1. Mouse megakafyocyteac0cultur.d with PA31742 cdla infactd with an amphotropk rmovirua. (A) A low powmview t o ahow
juxtepoakion of the peripheral zona (PZl of the megakaryocyte (MK) with the virua-packaging cell (PI on the right edge. Virua cannot be
seen at this magnification. (E) Another mature MK from a similar culture, a detail of which (area within rectangle) is ahown at higher
resolution in panel C. (CIViruses (arrows) are resotved in the demarcation membrane system. Original magnification: A, ~3,700; B,
~3,200;C. ~22,000.
PA317-N2 cells were plated on 60 mm tissue culture plates (Nunc;
V.W.R.. Piscataway. NJ) in 3 mL of Dulbecco's modified Eagles
medium (DMEM) supplemented with 10% (vol/vol) FCS and were
allowed to remain undisturbed for 24 hours, a t which time the plates
were approximately 70% confluent. Aliquots of 1 x IO6 murine
MK-enriched marrow cells were resuspended in 1.5 mL DMEM
supplemented with IO% FCS and 3 pg/mL of polybrene (Sigma. S t
Louis, MO). After the medium was aspirated from the plates
containing PA 317-N2, 1.5 mL of the marrow cell suspension was
added to each plate. Co-cultivation of PA317-N2 and murine MK
was allowed to proceed for 16 hours, after which time the cultures
were fixed in situ by addition of glutaraldehyde.
Infecrion o/ human MK wirh HIV. MK-enriched human marrow cells suspended in 0.5 mL a s described above were added to the
sedimented HIV-infected H9 cells to which, in some experiments.
platelets were added also. The specimens were incubated for 1, 2,
and 16 hours at 37OC with intermittent gentle manual agitation
during the first hour. Incubation was ended by the addition of cold
glutaraldehyde.
Elecrron microscopy. In the case of the murine samples. the
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ZUCUER-FRANKLIN. SERE-.
1922
specimens were dehydrated and embedded in situ in Mybed 812. as
dacriw.?The human "@e were d i m m l e d after fixation and
then embedded for clcctmn mkmsapv
, by rouiim mcthodr. Block
rarr, miaining w h r y o c y t a
WCm IckclCdin thick mionr.
Thin sections wmsiaimd with uranyl scctnte and l a d citrate. They
were viewed with an 1:lmiskg I c l a m mwosatpc a i inst"ent
magnificationsranging lrom 1O.ooO IO30.000.
RESULTS
M~~~~ meRcr~ar)my,es. buse
of their large
si,e,
mouse megakaryocyleswereeasilyidentified by phase micopy of thick sections. A$ shown in ~i~ I A , on electmn
microbcopy. the peripheral tone of the MK a p p c a d to be
loosely adherent to the suhrtrate of plastic-attached virus
packaging cells. but them was no noteworthy aberration in
M K morphology. The platelet fields and extensive develop
ment of the demarcation membrane system (DMS). characteristic for this specie. is secn clenrly in Fig 1R. At higher
resolution. interiorized virus particle were Ken with case
( i i p IC).In almost every instance. the particle appeared to
be located within thecisternae of the DMS and interior to the
peripheral tone of cytoplasm. Oarasionally. particles were
secn in membranebound spaces very close to the nucleus.
Fusion of virus envelop with MK membranes was not
observed, but mole exhaustive analyses will k necessary 10
-2.
AND ZHENG
determine whether this process occurs. Mouse MK 00.
c u l t u d with uninfmed PA 3 I7 cells did not -1
vim
particles.
Work with human megakaryocyta waseltremelydema,,&
ing. primarily because of the dearth of freshly isolated
normal human M K available and thevulnerabilityofthe cell
to ultrastructural damage as a consequence of in vitro
manipulation. However. there was little doubt that HIV is
also subject to interioritation by fully dificrentiated mature
M K (Fig 2A). Only a small percentage of human MK had
taken up HIV. in comparison with the number of mouse MK
that had intefiorited the amphotropic retrovirus. HIV particles were mostly seen in membrane bound Spa- that
Probably constituted pan Of the D W . Unfortunately. Wantilation Of the n u m k r Of Virum taken UP per cell or the
pemntage of MK that had taken up any virus was not
pmible. ~ U S the
C size relationships (MK. -100 to 120
rm: HIV. 80 10 100 nm:and thin sections. -60 nm) preclude
such quantitation.
Uptake of HIV by " a 1 human platelets was clearly
demonstrable (Fig 2R and C). but was far lcsscommofi than
we had expected on the basis of p i o u s studies with other
microorganismsand particulates."-" In theabscnoeofantiseN m to HW. it wasestimated that 1% or fewer platelet thin
-
-
~m(rdp).cJ.a~~rrkhm-eM.oad~oJlr(A)kcJId~M(rho*rhgm(rrorrr)rrkhkth.
k.n (Ird rrN). In tho pm0.n d hnrkrMng Mrv. Arrow
ypbr( Mrv4
or. n o n w i t h ohwm(d tho
mm.MT. We".IC)p(.w.t ahewhg
o c m u o k e o m . h h g n loom (a* Id.mMoblov*wa. A ) p h . q r * J . . t Q l o n d m k r o t W ( M T ) a w ~ ~ Irrtm. oILg*ulnugn(liat&n: A.
w
dwm(r d tho OMS. (0) Pl0tol.l Ch.1 hw
put#. In o m o a k ~ t u
nwdkm. Four pn&
x 7.000: 0. x 27.000: C. x 28.000.
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1923
UPTAKE OF RETROVIRUS BY MK AND PLATELETS
sections contained virus. Adding antiserum or prolonging the
incubation period resulted in platelet aggregation and ultrastructural damage that precluded interpretation.
DISCUSSION
The data presented here constitute the first report showing
incontrovertibly that retroviruses, including HIV-I, can be
taken up by mature megakaryocytes and platelets. Illustrations of mouse megakaryocytes containing Friend leukemia
virus were published 30 years ago," and the detrimental
effect on MK differentiation and platelet production was
well-recognized.'6 The thrombocytopenia associated with
cytomegalovirus infection of mice was also attributed primarily to its effect on megakaryocytes." In humans, studies
have been less definitive, but low megakaryocyte and platelet
counts are not uncommon in diseases of viral etiology, such as
measles and Dengue fever.'* To our knowledge, virus particles have not been delineated in bone marrow specimens
obtained from such patients. This also holds true for fresh
bone marrow isolated from patients with AIDS, although we
have shown previously that a large number of AIDS-MK
contain proviral RNA.6 What could be the significance of
viral uptake by fully differentiated MK and platelets? First,
because of the ability of the MK nucleus to undergo
endomitosis, integration of viral genes remains a possibility
throughout the lifespan of the cell. Second, denuded MK
nuclei as well as effete platelets are phagocytosed by macrophages and variants of these cells. Obviously, even an
occasional virus particle taken up by a circulating platelet
could contribute to the reservoir of HIV believed to exist in
the monocyte/macrophage phagocytic system.19."
The experiments conducted with mouse retrovirus were
designed to explore whether differentiated MK could still be
transfected with retrovirus-based genes defective or absent in
congenital platelet disorders. The observation that amphotropic retroviruses are avidly interiorized by MK is the first
positive step in this direction. Since excellent tissue culture
techniques for mouse megakaryocytes that culminate in
platelet production
this should be the first system in
which our hypothesis could be validated.
ACKNOWLEDGMENT
The technical assistance of George Grusky and Susan Dittmar is
gratefully acknowledged.
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manipulation of hematopoietic stem cells with retrovirus vectors.
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3. Seremetis S, Inghirami G, Ferrero D, Newcomb E, Knowles D,
Dotto G-P, Dalla-Favera R: Transformation and plasmacytoid
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Thailand. Ann Int Med 62367, 1965
19. Gartner S, Markovitz P, Markovitz DM, Kaplan MH, Gallo
RC, Popovic M: The role of mononuclear phagocytes in HTLV
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Fauci AS: Detection of AIDS virus in macrophages in brain tissue
from AIDS patients with encephalopathy. Science 233:1089, 1986
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1990 75: 1920-1923
Internalization of human immunodeficiency virus type I and other
retroviruses by megakaryocytes and platelets
D Zucker-Franklin, S Seremetis and ZY Zheng
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