Differential expression in human cells from the p6 promoter of human parvovirus B19 following plasmid transfection and recombinant adeno-associated virus 2 (AAV) infection: human megakaryocytic leukaemia cells are non-permissive for AAV infection.
Expression from the human parvovirus B19p6 promoter fused to the firefly luciferase ('Luc') reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, KB, and a human megakaryocytic leukaemia cell line, MB-02, known to become permissive for B19 replication following erythroid-differentiation. The B19p6-Luc construct was introduced into KB and MB-02 cells, both in undifferentiated and differentiated states, either via DNA-mediated transfection, or via infection with recombinant adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus known to possess a broad host-range. Although Luc activity was readily detected in KB cells following transfection of the B19p6-Luc plasmid DNA, no expression from the B19p6 promoter was observed following infection with recombinant virus. In addition, transfection of the reporter plasmid resulted in high-level expression of Luc in differentiated but not in undifferentiated MB-02 cells. However, no Luc activity was detected, even in differentiated MB-02 cells, following infection with recombinant virus. Further studies with an additional recombinant as well as wild-type (wt) AAV revealed that MB-02 cells were non-permissive for AAV infection. A second human megakaryocytic leukaemia cell line, M07e, was likewise resistant to infection by recombinant as well as wt AAV. Taken together, these studies identify the first human cell type that cannot be infected by AAV. They indicate that expression from the B19p6 promoter, in the context of an AAV genome, is restricted to primary human haematopoietic cells, perhaps because parvoviral DNA replication and transcription are intrinsically coupled.
Ponnazhagan
S
S
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
Wang
X S
XS
Woody
M J
MJ
Luo
F
F
Kang
L Y
LY
Nallari
M L
ML
Munshi
N C
NC
Zhou
S Z
SZ
Srivastava
A
A
D002454:Cell Differentiation; D003587:Cytomegalovirus / Q000502:physiology; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D007947:Leukemia, Megakaryoblastic, Acute; D008156:Luciferases / Q000096:biosynthesis; D016732:Parvovirus B19, Human / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D008156:Luciferases