Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events

Using a recently developed model for in vitro generation of pp65-positive p olymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immuno competent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA and immediate-early [IE] and pp67 late m RNAs) as early as 60 min after coculture with human umbilical vein endothel ial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains, The number of PMNLs positive for each viral parameter increased with coculture time. Usin g HELF infected with laboratory-adapted HCMV strains, only very small amoun ts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mR NA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls, Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE gen es were transcribed in PMNLs during coculture, Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedl y with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that t ransfer of infectious virus and viral products from infected cells to PMNLs Is likely to he mediated by microfusion events induced by wild-type strain s only. In addition, HCMV pp65 and p72 were both shown to localize in the n ucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild type strains only) of HUV EC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i,e,, endocytosis, o ccurs with both wild-type and laboratory strains and leads to the acquisiti on of very small amounts of viral nucleic acids. In conclusion, HCMV replic ates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, th us providing evidence for a potential mechanism of HCMV dissemination in vi vo.