[Inhibition of expression of CCR5 and CXCR4 on cells by adenovirus-mediated antisense RNA].
OBJECTIVE
To suppress the expression of CCR5 and CXCR4, the co-receptors for human immunodeficiency virus type 1 ( HIV-1), and thus inhibit HIV-1 from entering cells.


METHODS
DNA fragments encoding either CCR5 or CXCR4 were amplified from healthy human peripheral blood mononuclear cells (PBMCs) by reverse transcript polymerase chain reaction (RT-PCR) and sequencing was performed. Correct fragments were inserted into Shuttle plasmid inversely, which was recombined with backbone plasmid containing homologous adenoviral genome in E. coli BJ5183. The recombinant plasmids were transfected into 293 cells in which they were packaged and amplified. Recombinant adenoviruses containing antisense RNA of CCR5 or CXCR4 were obtained and identified by RT-PCR, and the titres of them were determined by cytopathic effect (CPE) method. The U937 and MT4 cells were infected by recombinant adenoviruses containing antisense RNA of CCR5 (multiplicity of infection, MOI = 100) and CXCR4 (MOI = 200), respectively. The expression of co-receptors on infected cell was measured by fluorescence activated cell sorter at 24, 48, 72 hours and 10 days after infection. In addition, the chemotactic activity and proliferation of infected cells were detected with Boyden chamber and 3H incorporation respectively.


RESULTS
We constructed the recombinant plasmids and obtained the recombinant adenoviruses which contained antisense RNA of CCR5 or CXCR4 and were designated as pAd-antiR5 and pAd-antiX4 respectively. The titers of recombinant adenoviruses pAd-antiR5 and pAd-antiX4 were 5 x 10" PFU/ml and 7 x 10(10) PFU/ml, respectively. The expression rate of CCR5 on U937 cells decreased from 82. 10% (blank control) to 1.12% (Ad-antiR5 infected) , and that of CXCR4 on MT4 cells decreased from 42% (blank control) to 1.03% (Ad-antiX4 infected) 24 hours later. The expression rates of CCR5 on Ad-antiR5 infected U937 cells were 1.02% , 1.26% , 1.23% at 48 hours, 72 hours, and 10 days later, respectively. The expression rates of CXCR4 on Ad-antiX4 infected MT4 cells were 1.13%, 1.17%, 1.22% at 48 hours, 72 hours, and 10 days later, respectively. Moreover, the recombinant adenovirus had no effects on chemotactic activity and proliferation of the cells.


CONCLUSIONS
The recombinant adenovirus containing antisense CCR5 or CXCR4 can remarkably decrease the expression of co-receptors for HIV-1 on U937 or MT4 cells without affecting their chemotactic activities and proliferative abilities.
Li
Wen-gang
WG
Department of Infectious Diseases, Peking University First Hospital, Beijing 100034, China.
Yu
Min
M
Bai
Li
L
Tian
Xiu-lan
XL
Zhang
Zheng
Z
Bu
Ding-fang
DF
Xu
Xiao-yuan
XY
D000256:Adenoviridae / Q000235:genetics; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D002633:Chemotaxis; D015536:Down-Regulation; D005822:Genetic Vectors; D006801:Humans; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D020298:U937 Cells
D016372:RNA, Antisense; D012333:RNA, Messenger; D019713:Receptors, CCR5; D019718:Receptors, CXCR4