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Productive Human Immunodeficiency Virus-1 Infection of Purified
Megakaryocytic Progenitors/Precursors and Maturing Megakaryocytes
C. Chelucci,
M. Federico,
R. Guerriero,
G. Mattia,
I. Casella,
E. Pelosi,
U. Testa,
G. Mariani,
H.J. Hassan, and
C. Peschle
From the Departments of Hematology-Oncology and Virology, Istituto
Superiore di Sanit, Rome, Italy; and Thomas Jefferson Cancer Institute,
Thomas Jefferson University, Philadelphia, PA.
We have evaluated the susceptibility to human immunodeficiency virus
(HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the
following approach: (1) human hematopoietic progenitor cells (HPCs),
stringently purified from peripheral blood and grown in serum-free
liquid suspension culture supplemented with thrombopoietin (Tpo),
generated a relatively large number of  98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3)
finally, the presence of viral mRNA and proteins was analyzed by
reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ
hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do
not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV
transcripts and proteins were clearly detected in all NL4-3 infection
experiments by RT-PCR and p24 assay, respectively, with the highest
viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs varies
from at least 1% and 5% for day 0 and day 5 infected cells,
respectively. Production of an infectious viral progeny, evaluated by
the capability of culture supernatants from day 5 NL4-3-challenged MKs
to infect C8166 T-lymphoblastoid cell line, was consistently observed
(viral titer,  5 × 103 tissue culture infectious
dose50/mL/106 cells). Exposure of
MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody
(MoAb), which recognizes the CD4 region binding with the gp120 envelope
glycoprotein, markedly inhibited HIV infection, as indicated by a
reduction of p24 content in the supernatants: because the inhibitory
effect was incomplete, it is apparent that the infection is only
partially CD4-dependent, suggesting that an alternative mechanism of
viral entry may exist. Morphologic analysis of day 12 MKs derived from
HPCs infected at day 0 showed an impaired megakaryocytic
differentiation/maturation: the percentage of mature MKs was markedly
reduced, in that 80% of cells showed only one nuclear lobe and a
pale cytoplasm with few granules. Conversely, megakaryocytic precursors
challenged at day 5 to 8 generated fully mature day 10 to 12 MKs
showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated
with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different
days of culture, showed an impaired megakaryocytopoiesis comparable to
that observed in HIV-infected cells, thus suggesting that Tat is a
major mediator in the above described phenomena. These results shed
light on the pathogenesis of HIV-related thrombocytopenia; furthermore,
they provide a model to investigate the effects of HIV on
megakaryocytic differentiation and function.
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1225-1234
© 1998 by The American Society of Hematology.
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