PRODUCTIVE HUMAN IMMUNODEFICIENCY VIRUS-1 INFECTION OF PURIFIED MEGAKARYOCYTIC PROGENITORS PRECURSORS AND MATURING MEGAKARYOCYTES/

We have evaluated the susceptibility to human immunodeficiency virus ( HIV)-1 infection of in vitro grown megakaryopoietic progenitors/predur sors and maturing megakaryocytes (MKs), based on the following approac h: (1) human hematopoietic progenitor cells (HPCs), stringently purifi ed from peripheral blood and grown in serum-free liquid suspension cul ture supplemented with thrombopoietin (Tpo), generated a relatively la rge number of greater than or equal to 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of cult ure (ie, 6, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multip licity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. o f the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymera se chain reaction (RT-PCR)/in situ hybridization and antigen capture a ssays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 Bat-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,. respectivel y, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs va ries from at least 1% and 5% for day 0 and day 5 infected cells, respe ctively. Production of an infectious viral progeny, evaluated by the c apability of culture supernatants from day 5 NL4-3-challenged MKs to i nfect C8166 T-lymphoblastoid cell line, was consistently observed (vir al titer, approximate to 5 x 10(3) tissue culture infectious doses(50) /mL/10(6) 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 i nfection, as indicated by a reduction of p24 content in the supernatan ts: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alte rnative mechanism of viral entry may exist. Morphologic analysis of da y 12 MKs derived from HPCs infected at day 0 showed an impaired megaka ryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that approximate to 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megak aryocytic precursors challenged at day 5 to 8 generated fully mature d ay 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhib itory effect of HIV on the megakaryopoietic gene program relates to th e differentiation stage of cells subjected to the viral challenge. Fin ally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, an alyzed at different days of culture, showed an impaired megakaryocytop oiesis comparable to that observed in HIV-infected cells, thus suggest ing that Tat is a major mediator in the above described phenomena. The se results shed light on the pathogenesis of HIV-related thrombocytope nia; furthermore, they provide a model to investigate the effects of H IV on megakaryocytic differentiation and function. (C) 1998 by The Ame rican Society of Hematology.