Suppression of normal human hematopoiesis by cytomegalovirus in vitro.

Human cytomegalovirus (HCMV) infection is associated with marrow suppression in immunocompromised patients. To examine the mechanism(s) underlying this suppression, the effect of a laboratory strain of HCMV (AD169) and a clinical isolate of HCMV on colony formation by normal human marrow (BMC) hematopoietic progenitors in the presence and/or absence of monocyte/macrophages (MO) and T cells was studied. Direct addition of HCMV at multiplicity of infection (MI) of 0.1 to 5 to BMC produced dose-dependent inhibition of colony forming unit-mix (CFU-Mix) (30-82%), CFU granulocyte, macrophage (CFU-GM) (15-98%), and burst forming unit-erythroid (BFU-E) (23-86%); no consistent effect of CFU-erythroid (CFU-E) was noted. This inhibition occurred both with the direct addition of HCMV to the culture plates as well as by the preincubation of BMC with HCMV followed by washing of the cells; significant inhibition (p < 0.01) of colony formation occurred after 1 hour of incubation at MI of 5. No suppression of colony formation occurred when UV-irradiated virus was used. The inhibitory effect of HCMV was reduced when MO and T cells were removed prior to exposure of marrow to virus at MI of 1 to 5. At MI of 20, however, HCMV suppressed colony formation by BMC depleted of MO and T cells by about 40%. Coculture of autologous or allogeneic T cells, but not MO, exposed to HCMV with intact or depleted marrow resulted in inhibition of CFU-Mix (51%), CFU-GM (40%), and BFU-E (37%). The inhibitory effect of virus-exposed T cells did not appear to be mediated through a soluble factor. T cells expressing CD8 antigen were most active in this process; natural killer (NK) cells were not active. These results suggest that the suppressive effect of HCMV on hematopoiesis may be mediated in part through T lymphocytes.

Characterization of a human cytomegalovirus 1.6-kilobase late mRNA and identification of its putative protein product.

In a previous study (J. Martinez, R. S. Lahijani, and S. C. St. Jeor, J. Virol. 63:233-241, 1989), we identified a late, unspliced 1.6-kb mRNA that maps to the HindIII R fragment of human cytomegalovirus (HCMV) AD169. In the present study, the direction of transcription of this mRNA was determined by Northern (RNA) analysis with strand-specific probes. Primer extension was used to precisely map the 5' end of the mRNA. An open reading frame (ORF) designated ORF 2-1, located 176 nucleotides downstream from the cap site of the 1.6-kb mRNA, was identified. A synthetic peptide was made representing a hydrophilic region in the amino terminus of ORF 2-1. Immunoprecipitation and Western immunoblot analysis of infected HEL cell lysates, using affinity-purified antibody to the peptide (anti-P2-1), detected a viral protein with an apparent molecular mass of 58 kDa late in infection. Further support for the presence of this protein in infected-cell lysates was obtained by an enzyme-linked immunosorbent assay. Expression of viral antigens in intact infected HEL cells was assessed by immunofluorescence. General cytoplasmic staining was observed at 62 h postinfection, in contrast to a localized staining observed in the nuclear and perinuclear region at 96 h postinfection.

Identification of a putative cell receptor for human cytomegalovirus.

Proteins from a variety of cell types were separated using SDS-PAGE, transferred to nitrocellulose filters, and probed with intact human cytomegalovirus (HCMV). Virus bound to cell proteins was detected directly using 125I-labeled HCMV or indirectly using an immunologic assay with a secondary antibody labeled with 125I. Using this approach proteins of molecular weights 32 and 34 kDa were identified that would bind HCMV and were present on T4+ and T8+ lymphocytes, a B lymphoblastoid cell line, and human diploid fibroblasts. Binding of labeled virus to cells could be blocked by the addition of unlabeled homologous virus. Treatment of virus with NP40 to remove the virus envelope blocked binding to cellular proteins. Both the 32- and 34-kDa proteins could be copurified with cellular membranes. HCMV bound equally well to T4+ and T8+ cells as well as to either replicating (PHA or interleukin-2 stimulated) or resting lymphocytes. Interestingly, there appeared to be a single binding site for HCMV on human fibroblasts (34 kDa). The results support the idea that there is a receptor for HCMV present on the surface of human lymphocytes and fibroblasts.