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 polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial 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. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, 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 genes 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 markedly 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 transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus 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 HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.
Gerna
G
G
Servizio di Virologia, Area Infettivologica, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy. g.gerna@smatteo.pv.it
Percivalle
E
E
Baldanti
F
F
Sozzani
S
S
Lanzarini
P
P
Genini
E
E
Lilleri
D
D
Revello
M G
MG
D002459:Cell Fusion / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology; D002478:Cells, Cultured; D018920:Coculture Techniques; D003513:Cycloheximide / Q000494:pharmacology; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D003609:Dactinomycin / Q000494:pharmacology; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure / Q000821:virology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D017245:Foscarnet / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000166:cytology / Q000196:embryology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure / Q000821:virology*; D014176:Protein Biosynthesis / Q000187:drug effects; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014471:Umbilical Veins / Q000166:cytology; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics; D019562:Viral Load; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis; D014779:Virus Replication / Q000187:drug effects
D004279:DNA, Viral; D012367:RNA, Viral; D019010:Vascular Cell Adhesion Molecule-1; D014764:Viral Proteins; D003609:Dactinomycin; D017245:Foscarnet; D003513:Cycloheximide